Detection of Chlamydia and Chlamydia-like organisms in bovine placental tissue.

This study aimed to investigate the presence of Chlamydia spp. and Parachlamydia acanthamoebae in bovine placental tissue originating from abortion and non-abortion cases in Belgium. Placentas of 164 late term bovine abortions (last trimester of gestation) and 41 non-abortion (collected after calving) cases were analysed by PCR for Chlamydia spp., Chlamydia abortus, C. psittaci and P. acanthamoebae. Additionally, a subset of 101 (75 abortion and 26 non-abortion cases) of these placenta samples were also analysed by histopathology to detect possible Chlamydia-induced lesions. In 5.4% (11/205) of the cases, Chlamydia spp. were detected, and three of those cases were positive for C. psittaci. Parachlamydia acanthamoebae was detected in 36% (75/205) of the cases, being 44% (n = 72) in abortions and 7.3% (n = 3) in non-abortions cases (p < .001). None of the cases was positive for C. abortus. Purulent and/or necrotizing placentitis with or without vasculitis was observed in 18.8% (19/101) of the histopathologically analysed placenta samples. In 5.9% (6/101) of the cases, placentitis was observed along with vasculitis. In the abortion cases, 24% (18/75) of the samples showed purulent and/or necrotizing placentitis, while purulent and/or necrotizing placentitis was visible in 3.9% (1/26) of the non-abortion cases. Placental lesions of inflammation and/or necrosis were present in 44% (15/34) of the cases where P. acanthamoebae was detected, while inflammation and/or necrosis was present in 20.9% (14/67) of the negative cases (p < .05). The detection of Chlamydia spp. and especially P. acanthamoebae, in combination with correlated histological lesions such as purulent and/or necrotizing placentitis and/or vasculitis in placental tissue following abortion, suggests a potential role of this pathogen in cases of bovine abortion in Belgium. Further in-depth studies are necessary to unravel the role of these species as abortifacient agents in cattle and to include them in bovine abortion monitoring programmes.

Fetoplacental pathology of equine abortion, premature birth, and neonatal loss due to Chlamydia psittaci.

This report describes the fetoplacental pathology of Chlamydia psittaci-associated abortion, premature birth, and neonatal loss in 46 of 442 equine abortion investigations between 2015 and 2019. Seven abortions, 26 premature births, and 13 neonatal deaths with positive C. psittaci polymerase chain reaction (PCR) were evaluated. In 83% of cases (38/46), C. psittaci infection was considered as the primary cause of loss based on quantitative PCR (qPCR) confirmation, pathological findings, and exclusion of other causes, and was supported by Chlamydia spp immunolabeling in fetoplacental lesions. Lymphohistiocytic placentitis with vasculitis (36/38) affected the amnion, umbilical cord, and chorioallantois at the umbilical vessel insertion and/or cervical pole. Lymphohistiocytic chorionitis in the subvillous stroma extended to the allantois mostly without villous destruction. Lymphohistiocytic amnionitis and funisitis occurred at the amniotic cord attachment. Lymphohistiocytic hepatitis was observed in 19/38 cases and pneumonia was identified in 26 cases. Chlamydia spp immunolabeled in placenta, lung, liver, or splenic tissue in the cases that were tested (14/38). C. psittaci infection was not the cause of loss in 2 cases with other diseases and of uncertain significance in 6 cases with no conclusive cause of loss. immunohistochemistry (IHC) was negative for 6 of these cases (6/8). The highest Chlamydia load was detected in pooled placental tissues by qPCR. qPCR and IHC had 83% congruence at a qPCR cut-off of 1 gene copy. IHC limits of detection corresponded to infections with 2 × 102 gene copies identified by qPCR. This study confirms the etiological role of C. psittaci as a cause of naturally occurring equine reproductive loss.

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.

Intestinal Goblet Cell Loss during Chorioamnionitis in Fetal Lambs: Mechanistic Insights and Postnatal Implications.

Chorioamnionitis, an important cause of preterm birth, is linked to necrotizing enterocolitis (NEC). NEC is characterized by a disrupted mucus barrier, goblet cell loss, and endoplasmic reticulum (ER) stress of the intestinal epithelium. These findings prompted us to investigate the mechanisms underlying goblet cell alterations over time in an ovine chorioamnionitis model. Fetal lambs were intra-amniotically (IA) exposed to lipopolysaccharides (LPS) for 5, 12, or 24 h, or 2, 4, 8, or 15 d before premature delivery at 125 d gestational age (GA). Gut inflammation, the number, distribution, and differentiation of goblet cells, ER stress, and apoptosis were measured. We found a biphasic reduction in goblet cell numbers 24 h-2 d after, and 15 d after IA LPS exposure. The second decrease of goblet cell numbers was preceded by intestinal inflammation, apoptosis, and crypt ER stress, and increased SAM-pointed domain-containing ETS transcription factor (SPDEF)-positive cell counts. Our combined findings indicated that ER stress drives apoptosis of maturating goblet cells during chorioamnionitis, ultimately reducing goblet cell numbers. As similar changes have been described in patients suffering from NEC, these findings are considered to be clinically important for understanding the predecessors of NEC, and targeting ER stress in this context is interesting for future therapeutics.

Chorioamnionitis induces enteric nervous system injury: effects of timing and inflammation in the ovine fetus.

BACKGROUND: Chorioamnionitis, inflammation of the chorion and amnion, which often results from intrauterine infection, is associated with premature birth and contributes to significant neonatal morbidity and mortality, including necrotizing enterocolitis (NEC). Recently, we have shown that chronic chorioamnionitis is associated with significant structural enteric nervous system (ENS) abnormalities that may predispose to later NEC development. Understanding time point specific effects of an intra-amniotic (IA) infection on the ENS is important for further understanding the pathophysiological processes and for finding a window for optimal therapeutic strategies for an individual patient. The aim of this study was therefore to gain insight in the longitudinal effects of intrauterine LPS exposure (ranging from 5 h to 15 days before premature delivery) on the intestinal mucosa, submucosa, and ENS in fetal lambs by use of a well-established translational ovine chorioamnionitis model. METHODS: We used an ovine chorioamnionitis model to assess outcomes of the fetal ileal mucosa, submucosa and ENS following IA exposure to one dose of 10 mg LPS for 5, 12 or 24 h or 2, 4, 8 or 15 days. RESULTS: Four days of IA LPS exposure causes a decreased PGP9.5- and S100ß-positive surface area in the myenteric plexus along with submucosal and mucosal intestinal inflammation that coincided with systemic inflammation. These changes were preceded by a glial cell reaction with early systemic and local gut inflammation. ENS changes and inflammation recovered 15 days after the IA LPS exposure. CONCLUSIONS: The pattern of mucosal and submucosal inflammation, and ENS alterations in the fetus changed over time following IA LPS exposure. Although ENS damage seemed to recover after prolonged IA LPS exposure, additional postnatal inflammatory exposure, which a premature is likely to encounter, may further harm the ENS and influence functional outcome. In this context, 4 to 8 days of IA LPS exposure may form a period of increased ENS vulnerability and a potential window for optimal therapeutic strategies.

Steroid synthesis and metabolism in the equine placenta during placentitis.

Equine placentitis is associated with alterations in maternal peripheral steroid concentrations, which could negatively affect pregnancy outcome. This study aimed to elucidate the molecular mechanisms related to steroidogenesis and steroid-receptor signaling in the equine placenta during acute placentitis. Chorioallantois (CA) and endometrial (EN) samples were collected from mares with experimentally induced placentitis (n = 4) and un-inoculated gestationally age-matched mares (control group; n = 4). The mRNA expression of genes coding for steroidogenic enzymes (3ßHSD, CYP11A1, CYP17A1, CYP19A1, SRD5A1, and AKR1C23) was evaluated using qRT-PCR. The concentration of these enzyme-dependent steroids (P5, P4, 5αDHP, 3αDHP, 20αDHP, 3ß-20αDHP, 17OH-P, DHEA, A4, and estrone) was assessed using liquid chromatography-tandem mass spectrometry in both maternal circulation and placental tissue. Both SRD5A1 and AKR1C23, which encode for the key progesterone metabolizing enzymes, were downregulated (P < 0.05) in CA from the placentitis group compared to controls, and this downregulation was associated with a decline in tissue concentrations of 5αDHP (P < 0.05), 3αDHP (P < 0.05), and 3ß-20αDHP (P = 0.052). In the EN, AKR1C23 was also downregulated in the placentitis group compared to controls, and this downregulation was associated with a decline in EN concentrations of 3αDHP (P < 0.01) and 20αDHP (P < 0.05). Moreover, CA expression of CYP19A1 tended to be lower in the placentitis group, and this reduction was associated with lower (P = 0.057) concentrations of estrone in CA. Moreover, ESR1 (steroid receptors) gene expression was downregulated (P = 0.057) in CA from placentitis mares. In conclusion, acute equine placentitis is associated with a local withdrawal of progestins in the placenta and tended to be accompanied with estrogen withdrawals in CA.

Pregnancy loss due to amnionitis in anglo-arabian mare-Case report.

The amnion is a layer of the foetal membrane that has mechanical protection function and allows expansion and growth. The diseases that affect amnion have rarely been reported in horses. Amnionitis and funiculitis have been implicated as components of abortions as observed in abortions caused by the mare reproductive loss syndrome. Here, we report an abortion at 240-day gestation of a 7-year-old Anglo-Arabian mare with a history of previous stillbirth. Blood samples from both mare and foetus were collected for serological testing. Fragments from the placenta were taken for macroscopic and histopathological examinations. The results of these tests ruled out leptospirosis and concluded that the cause of the abortion was non-infectious chronic amnionitis. Moreover, the decreased vascularization in the placenta was consistent with placental insufficiency. This case highlights the importance of monthly ultrasound monitoring in equine pregnancy in order to diagnose placental insufficiency. The study also confirmed the efficiency of the histopathological examination for the definitive diagnosis of placental inflammation and for the study of foetal vascularization to rule out placental insufficiency in equine reproduction.

Equine placentitis is associated with a downregulation in myometrial progestin signaling†.

The current study aimed to elucidate the mechanisms underlying myometrial activation during equine placentitis related to progestogens and the progesterone receptor signaling pathways. Placentitis was induced via intracervical inoculation with Streptococcus equi ssp zooepidemicus in mares at approximately 290 days of gestation (placentitis group; n = 6) with uninoculated gestationally matched mares as controls (n = 4). Mares in the placentitis and control groups were euthanized, and myometrial samples were collected from two regions: region 1-parallel to active placentitis lesion with placental separation in placentitis group (P1) or caudal pole of the placenta in control group (C1); and region 2-parallel to apparently normal placenta without separation in placentitis group (P2) or uterine body in control group (C2). In the current study, SRD5A1 and AKR1C23, which encode for the key P4 metabolizing enzymes, were downregulated in P1 in comparison to C1, C2, and P2, and this was associated with a decline (P < 0.05) in 5αDHP, allopregnanolone (3αDHP), and 20αDHP in P1 in comparison to C1. Further, myometrial expression of PR was downregulated (P < 0.05) in P1 in comparison to C1 and P2, and this was associated with activation of the inflammatory cascade as reflected by significant upregulation of IL-1ß and IL-8 in P1 in comparison to C1, C2, and P2, and supported by increased tissue leukocytes in P1 in comparison to C1. In conclusion, equine placentitis is associated with a localized withdrawal of progestins and a downregulation of the PR in the myometrium concomitant with upregulation of inflammatory cytokines and subsequent myometrial activation.

The urticating setae of Ochrogaster lunifer, an Australian processionary caterpillar of veterinary importance.

The bag-shelter moth, Ochrogaster lunifer Herrich-Schaffer (Lepidoptera: Notodontidae), is associated with a condition called equine amnionitis and fetal loss (EAFL) on horse farms in Australia. Setal fragments from O. lunifer larvae have been identified in the placentas of experimentally aborted fetuses and their dams, and in clinical abortions. The gregarious larvae build silken nests in which large numbers cohabit over spring, summer and autumn. The final instars disperse to pupation sites in the ground where they overwinter. Field-collected O. lunifer larvae, their nests and nearby soil were examined using light and electron microscopy to identify setae likely to cause EAFL and to determine where and how many were present. Microtrichia, barbed hairs and true setae were found on the exoskeletons of the larvae. True setae matching the majority of setal fragments described from equine tissue were found on third to eighth instar larvae or exuviae. The number of true setae increased with the age of the larva; eighth instars carried around 2.0-2.5 million true setae. The exuvia of the pre-pupal instar was incorporated into the pupal chamber. The major sources of setae are likely to be nests, dispersing pre-pupal larvae and their exuviae, and pupal chambers.

Ureaplasma parvum undergoes selection in utero resulting in genetically diverse isolates colonizing the chorioamnion of fetal sheep.

Ureaplasmas are the microorganisms most frequently isolated from the amniotic fluid of pregnant women and can cause chronic intrauterine infections. These tiny bacteria are thought to undergo rapid evolution and exhibit a hypermutatable phenotype; however, little is known about how ureaplasmas respond to selective pressures in utero. Using an ovine model of chronic intraamniotic infection, we investigated if exposure of ureaplasmas to subinhibitory concentrations of erythromycin could induce phenotypic or genetic indicators of macrolide resistance. At 55 days gestation, 12 pregnant ewes received an intraamniotic injection of a nonclonal, clinical Ureaplasma parvum strain followed by (i) erythromycin treatment (intramuscularly, 30 mg/kg/day, n = 6) or (ii) saline (intramuscularly, n = 6) at 100 days gestation. Fetuses were then delivered surgically at 125 days gestation. Despite injecting the same inoculum into all the ewes, significant differences between amniotic fluid and chorioamnion ureaplasmas were detected following chronic intraamniotic infection. Numerous polymorphisms were observed in domain V of the 23S rRNA gene of ureaplasmas isolated from the chorioamnion (but not the amniotic fluid), resulting in a mosaiclike sequence. Chorioamnion isolates also harbored the macrolide resistance genes erm(B) and msr(D) and were associated with variable roxithromycin minimum inhibitory concentrations. Remarkably, this variability occurred independently of exposure of ureaplasmas to erythromycin, suggesting that low-level erythromycin exposure does not induce ureaplasmal macrolide resistance in utero. Rather, the significant differences observed between amniotic fluid and chorioamnion ureaplasmas suggest that different anatomical sites may select for ureaplasma subtypes within nonclonal, clinical strains. This may have implications for the treatment of intrauterine ureaplasma infections.

Processionary caterpillar setae and equine fetal loss: 1. Histopathology of experimentally exposed pregnant mares.

Six pregnant Standardbred mares aged between 6 and 14 years were gavaged with 50 g or 100 g of suspended emulsified whole Processionary caterpillars (Ochrogaster lunifer) for 5 days during 2 experiments undertaken to study the etiology of equine amnionitis and fetal loss (EAFL). The 6 treated mares and 1 untreated mare were between 128 and 252 days gestation. Mare 1 (untreated) was euthanized on day 5 of the treatment period, while the treated mares were euthanized on days 2, 4, 8, 10, 12, and 24 days from their first treatment. Caterpillar setae were not found in the untreated mare. Setal fragments were present in all regions of the gastrointestinal tracts in all treated mares, the uteri and mesenteric lymph nodes of 5 mares, and the liver of 4 mares. Acute gastroenteritis of varying severity was present in all treated mares, and 5 of 6 mares had acute colitis and endometritis. Focal hyperplastic serositis was found in the duodenum, cecum, dorsal colon, and uteri of various mares occasionally with embedded setal fragments. Setal invasion of the mucosa evoked a range of lesions including superficial erosion to deep ulceration. Inflammation in deeper tissues ranged from unapparent to neutrophilic (microabscesses), eosinophilic, or mononuclear (microgranulomas). The finding of setal fragments within the uterus of experimental mares suggests that direct migration of setal fragments acting as a bacterial vector is a likely mechanism for the bacterial abortions that characterize equine amnionitis and fetal loss.

Processionary caterpillar setae and equine fetal loss: 2. Histopathology of the fetal-placental unit from experimentally exposed mares.

Pregnant mares were experimentally exposed to whole caterpillar or exoskeleton of the Processionary caterpillar (Ochrogaster lunifer) via gavage. Tissues were collected from resulting abortions and near or full-term pregnancies consisting of 13 aborted fetuses, 3 fetuses from treated euthanized mares, membranes of 5 foals, and organs from 3 foals. Three control membranes and 1 control fetus and membranes were examined. Caterpillar setal fragments were present in the allantochorion of the 3 fetuses from the euthanized mares and 11 of 12 aborted fetuses (92%) embedded in the chorion (villi or stroma) or allantois (vasculature or stroma). Placental locations of fragments ranged from the cervical pole region to the body encompassing the umbilical insertion and pregnant horn. Numbers in each fetus ranged from 1 to 7 fragments. Setae were present in the allantochorion from 2 to 22 days after the initial treatment. Acute to chronic active inflammation was present in all aborted fetuses, all euthanized fetuses, and within at least 1 tissue level (chorion, allantois, umbilical cord, or amnion) of the membranes from full-term foals. Amnionitis, funisitis, and allantoitis were present in 95% of the examined membranes. Pneumonia was present in 95% of the specimens, and bacteria were present histologically in 90.5% of the specimens with or without accompanying inflammation. The rapid migration of setae within 2 days after mare exposure suggests that direct setal migration into the fetal membranes is a likely initiating factor for equine amnionitis and fetal loss (EAFL).

IL-1 and TNF mediates IL-6 signaling at the maternal-fetal interface during intrauterine inflammation.

Introduction: IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP). Methods: Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation. Results: IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling. Discussion: These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

Bacterial infection in an Irrawaddy dolphin (Orcaella brevirostris).

One pregnant captive Irrawaddy dolphin (Orcaella brevirostris), with a body length of 225 cm, was found dead on 8 June 2009. The dolphin was anorexic and circling at the bottom of the pool before death. Laboratory tests revealed an increased leukocyte count and decreased platelet count; increased erythrocyte sedimentation rate; and slightly decreased red blood cell count, hemoglobin, and hematocrit. Alkaline phosphatase, creatinine, and glucose were significantly decreased. Moreover, uric acid and alanine aminotransferase and aspartate aminotransferase levels were elevated. A 57-cm fetus was recovered. The respiratory system, intestinal mucosa, mesentery and mesenteric lymph nodes, and spleen were congested and hemorrhagic. The heart, liver, and kidney appeared normal. Klebsiella spp. and Staphylococcus aureus were identified in the amniotic fluid. This is the first case report of bacterial infection in an Irrawaddy dolphin.

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.

Morphometric analysis of Hofbauer cells in normal placenta and chorioamnionitis in humans.

Hofbauer cells are macrophages residing in the stroma of placental villi and play a number of roles during normal pregnancy, as well as pathological conditions. A morphometric analysis of Hofbauer cells, in particular to investigate the number of cells, their size and shape in samples of normal human placenta from 1st trimester, term and with chorioamnionitis was performed. Tissue samples were immunostained for CD206 antigen and evaluated using ImageJ software. We detected significant changes in number and morphology of HBCs between normal placenta and placenta with chorioamnionitis samples. In chorioamnionitis, the cells were unevenly distributed within the villi, generally present in higher numbers, larger and more elongated than those in normal 1st trimester and term placenta.

Kinetics of placenta-specific 8 (PLAC8) in equine placenta during pregnancy and placentitis.

Placenta-specific 8 (PLAC8) is one of the placenta-regulatory genes which is highly conserved among eutherian mammals. However, little is known about its expression in equine placenta (chorioallantois; CA and endometrium; EN) during normal and abnormal pregnancy. Therefore, the current study was designed to 1) elucidate the expression of PLAC8 in equine embryonic membranes during the preimplantation period, 2) characterize the expression profile of PLAC8 in equine CA (45d, 4mo, 6mo, 10 mo, 11 mo and postpartum) and EN (14d, 4mo, 6mo, 10 mo, and 11 mo) obtained from pregnant mares (n = 4/timepoint), as well as, d14 non-pregnant EN (n = 4), and 3) investigate the expression profile of PLAC8 in ascending placentitis (n = 5) and in nocardioform placentitis (n = 6) in comparison to normal CA. In the preimplantation period, PLAC8 mRNA was not abundant in the trophectoderm of d8 equine embryo and d14 conceptus, while it was abundant later in d 30, 31, 34, and 45 chorion. In normal pregnancy, PLAC8 mRNA expression in CA at 45 d gradually decline to reach nadir at 6mo before gradually increasing to its peak at 11mo and postpartum CA. The mRNA expression of PLAC8 was significantly upregulated in CA from mares with ascending and nocardioform placentitis compared to control mares. Immunohistochemistry revealed that PLAC8 is localized in equine chorionic epithelium and immune cells. Our results revealed that PLAC8 expression in equine chorion is dynamic during pregnancy and is regulated in an implantation-dependent manner. Moreover, PLAC8 is implicated in the immune response in CA during equine ascending placentitis and 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.

Pulmonary vascular and alveolar development in preterm lambs chronically colonized with Ureaplasma parvum.

Ureaplasma species, the most commonly isolated microorganisms in women with chorioamnionitis, are associated with preterm delivery. Chorioamnionitis increases the risk and severity of bronchopulmonary dysplasia and persistent pulmonary hypertension in newborns. It is not known whether the timing of exposure to inflammation in utero is an important contributor to the pathogenesis of bronchopulmonary dysplasia. We hypothesized that chronic inflammation would alter the pulmonary air space and vascular development after 70 days of exposure to infection. Pregnant ewes were given intra-amniotic injection of Ureaplasma parvum serovars 3 or 6 at low (2 x 10(4) cfu) or high doses (2 x 10(7) cfu) or media (controls) at 55 days gestational age. Fetuses were delivered at 125 days (term = 150 days). U. parvum was grown from the lungs of all exposed fetuses, and neutrophils and monocytes were increased in the air spaces. Lung mRNA expression of IL-1beta and IL-8, but not IL-6, was modestly increased in U. parvum-exposed fetuses. U. parvum exposure increased surfactant and improved lung gas volumes. The changes in lung inflammation and maturation were independent of serovar or dose. Exposure to U. parvum did not change multiple indices of air space or vascular development. Parenchymal elastin and collagen content were similar between groups. Expression of several endothelial proteins and pulmonary resistance arteriolar media thickness were also not different between groups. We conclude that chronic exposure to U. parvum does not cause sustained effects on air space or vascular development in premature lambs.

Fibrinonecrotic Placentitis and Abortion Associated With Pantoea agglomerans Infection in a Mare.

Pantoea agglomerans, family Enterobacteriaceae, is a Gram-negative bacterium that may be isolated from soil and from plants. This bacterium has been associated with disease in plants, humans, and rarely in domestic animal species. We describe here a case of fibrinonecrotic placentitis and equine abortion associated with P. agglomerans infection in southern Brazil. A fetus with 10 months of gestation and its placenta were evaluated. Gross lesions were observed in the cervical star extending to the body of the chorioallantois and consisted of a focally extensive, transmural, severely thickened yellow area. Histologically, this area in the chorioallantois was effaced by severe necrosis, associated with marked inflammatory infiltrate of neutrophils and abundant deposition of fibrin and cellular debris. Aggregates of bacterial rods were noted intermixed with inflammation areas. No significant lesions were observed in the remaining organs inspected. Tissue samples of the lung, placenta, and stomach contents were cultured, and microbiological tests revealed the growth of P. agglomerans in all evaluated samples. The present study reaffirms the participation of P. agglomerans as a cause of bacterial placentitis and abortion in horses.