A DIO2 missense mutation and its impact on fetal response to PRRSV infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection. Our objectives were to identify mutation(s) within the porcine DIO2 gene and to determine if they were associated with fetal outcomes after PRRSV-2 challenge. Sequencing of the DIO2, genotyping identified variants, and association of DIO2 genotypes with fetal phenotypes including DIO2 mRNA levels, viability, survival, viral loads, cortisol and thyroid hormone levels, and growth measurements were conducted. RESULTS: A missense variant (p.Asn91Ser) was identified in the parental populations from two independent PRRSV-2 challenge trials. This variant was further genotyped to determine association with fetal PRRS outcomes. DIO2 mRNA levels in fetal heart and kidney differed by the genotypes of Asn91Ser substitution with significantly greater DIO2 mRNA expression in heterozygotes compared with wild-type homozygotes (P < 0.001 for heart, P = 0.002 for kidney). While Asn91Ser did not significantly alter fetal viability and growth measurements, interaction effects of the variant with fetal sex or trial were identified for fetal viability or crown rump length, respectively. However, this mutation was not related to dysregulation of the hypothalamic-pituitary-adrenal and thyroid axis, indicated by no differences in circulating cortisol, T4, and T3 levels in fetuses of the opposing genotypes following PRRSV-2 infection. CONCLUSIONS: The present study suggests that a complex relationship among DIO2 genotype, DIO2 expression, fetal sex, and fetal viability may exist during the course of fetal PRRSV infection. Our study also proposes the increase in cortisol levels, indicative of fetal stress response, may lead to fetal complications, such as fetal compromise, fetal death, or premature farrowing, during PRRSV infection.

Effect of porcine reproductive and respiratory syndrome virus 2 on angiogenesis and cell proliferation at the maternal-fetal interface.

Angiogenesis and cell proliferation in reproductive tissues are essential events for the maintenance of pregnancy, and alterations can lead to compromised fetal development and survival. Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) induces reproductive disease with negative financial and production impact on the swine industry. PRRSV-2 infection alters placental physiology through inflammatory and apoptotic pathways, yet fetal susceptibility varies. This study aimed to evaluate angiogenesis and cell proliferation in the porcine maternal-fetal interface (MFI) and determine if these physiological processes were altered by PRRSV-2 infection. Thirty-one pregnant gilts were inoculated with PRRSV-2 at gestation day 86 ± 0.4 (mean ± SD). Seven control gilts were sham-inoculated. All gilts were euthanized at 12 days postinoculation. Angiogenesis and cell proliferation were determined through the detection of vascular endothelial growth factor (VEGF) and Ki-67, respectively, using immunofluorescence of the MFI from 4 fetal resilience groups: uninfected (UNIF), high viral load-viable (HVL-VIA), and HVL-meconium-stained (MEC) from PRRSV-infected gilts, as well from sham-inoculated (CON) gilts. VEGF immunolabeling in the uterine submucosa was significantly lower in MEC compared with UNIF and HVL-VIA groups. Significantly greater Ki67 immunolabeling was detected in the trophoblasts of CON fetuses versus all other groups, and in uterine epithelium of CON and UNIF fetuses versus HVL-VIA and MEC. These results suggest that fetal resilience may be related to greater cell proliferation in uterine epithelium, and fetal compromise with reduced uterine submucosal angiogenesis, except fetuses with intrauterine growth restriction, in which inherently lower submucosal angiogenesis may be protective against PRRSV infection.

Fetal hypoxia and apoptosis following maternal porcine reproductive and respiratory syndrome virus (PRRSV) infection.

BACKGROUND: Mechanisms of fetal death following maternal PRRSV2 infection remain uncharacterized, although hypoxia from umbilical cord lesions and/or placental detachment due to apoptosis are hypothesized. We performed two experiments examining hypoxia and apoptosis in PRRSV-infected and non-infected, third-trimester fetuses to elucidate possible associations with fetal death. Fetuses were selected based on four phenotypic infection groups: fetuses from non-challenged control gilts (CTRL); low viral load fetuses (LVL; Exp 1) or uninfected fetuses (UNINF; Exp 2) from inoculated gilts; viable high viral load fetuses (HVL-VIA); and HVL meconium-stained fetuses (HVL-MEC). RESULTS: In experiment 1, paraffin embedded fetal tissues collected 21 days post maternal infection (DPI) were examined for DNA fragmentation associated with apoptosis. Positively stained foci were larger and more numerous (P < 0.05) in heart, liver, and thymus of HVL-VIA and HVL-MEC compared to CTRL and LVL fetuses. In experiment 2, group differences in gene expression within the hypoxia (HIF1a, IDO1, VEGFa, LDHA, NOS2, NOX1) and apoptosis (CASP3, CASP7, CASP8, CASP9, RIPK1, RIPK3) pathways were assessed by RT-qPCR in fetal tissues collected at 12 DPI. High viral load fetuses showed differential expression relative to the CTRL and UNINF (P < 0.05 for all). Brain tissue from HVL-VIA and HVL-MEC fetuses presented increased expression of CASP7, CASP8, RIPK3, HIF1a and IDO1. Fetal heart showed increased expression of CASP8, HIF1a, IDO and NOX1 and a decrease in NOS2 expression in infected groups. CASP7, CASP9, RIPK1 and RIPK3 were only increased in the heart of HVL-VIA while VEGFa was only increased for HVL-MEC fetuses. Thymus from HVL-MEC had decreased expression of CASP9 and there was increased IDO1 in all infected fetuses. CONCLUSIONS: There is strong evidence of apoptosis occurring in the heart, liver and thymus of highly viral load fetuses at 21 DPI. Furthermore, there was clear upregulation of apoptotic genes in the heart of high viral load infected fetuses and less prominent upregulation in the brain of PRRSV-infected fetuses, whereas thymus appears to be spared at 12 DPI. There was no strong evidence of hypoxia at 12 DPI in brain and thymus but some indication of hypoxia occurring in fetal heart.

Differential responses in placenta and fetal thymus at 12 days post infection elucidate mechanisms of viral level and fetal compromise following PRRSV2 infection.

BACKGROUND: A pregnant gilt infected with porcine reproductive and respiratory syndrome virus (PRRSV) can transmit the virus to her fetuses across the maternal-fetal-interface resulting in varying disease outcomes. However, the mechanisms leading to variation in fetal outcome in response to PRRSV infection are not fully understood. Our objective was to assess targeted immune-related gene expression patterns and pathways in the placenta and fetal thymus to elucidate the molecular mechanisms involved in the resistance/tolerance and susceptibility of fetuses to PRRSV2 infection. Fetuses were grouped by preservation status and PRRS viral load (VL): mock infected control (CTRL), no virus detected (UNINF), virus detected in the placenta only with viable (PLCO-VIA) or meconium-stained fetus (PLCO-MEC), low VL with viable (LVL-VIA) or meconium-stained fetus (LVL-MEC), and high VL with viable (HVL-VIA) or meconium-stained fetus (HVL-MEC). RESULTS: The host immune response was initiated only in fetuses with detectable levels of PRRSV. No differentially expressed genes (DEG) in either the placenta or thymus were identified in UNINF, PLCO-VIA, and PLCO-MEC when compared to CTRL fetuses. Upon fetal infection, a set of core responsive IFN-inducible genes (CXCL10, IFIH1, IFIT1, IFIT3, ISG15, and MX1) were strongly upregulated in both tissues. Gene expression in the thymus is a better differentiator of fetal VL; the strong downregulation of several innate and adaptive immune pathways (e.g., B Cell Development) are indicative of HVL. Gene expression in the placenta may be a better differentiator of fetal demise than the thymus, based-on principle component analysis clustering, gene expression patterns, and dysregulation of the Apoptosis and Ubiquitination pathways. CONCLUSION: Our data supports the concept that fetal outcome in response to PRRSV2 infection is determined by fetal, and more significantly placental response, which is initiated only after fetal infection. This conceptual model represents a significant step forward in understanding the mechanisms underpinning fetal susceptibility to the virus.

Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes.

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

Fetal cytokine response to porcine reproductive and respiratory syndrome virus-2 infection.

To understand the fetal immune response to porcine reproductive and respiratory virus-2 (PRRSV) and to evaluate the association with fetal viability, pregnant gilts were challenged on gestation day 85 and euthanized 21 days post infection. Based on preservation status and viral load in serum and thymus, fetuses were classified as either uninfected-viable (UNIF), high viral load viable (HV-VIA), or high viral load meconium stained (HV-MEC) and were compared with age matched control (CON) fetuses derived from mock infected gilts. Gene expression of IFNB, IFNG, CCL2, CCL5, CXCL10 and IL10, were all found to be significantly upregulated in the thymus and spleen of both high viral load groups. UNIF fetuses remained largely unaffected, with only small upregulations in IFNA and IL10 in the thymus, and IFNA, CCL5 and CXCL10 in the spleen. Regarding fetal viability, expression of CCL5 was significantly elevated in the thymus and spleen of HV-MEC compared to HV-VIA fetuses. The concentrations of IFNα, IFNγ, TNFα and CCL2 were elevated in the sera of all infected fetuses, whereas IFNß was below the detection limit in all fetal sera. Additional gene expression analysis in the thymus showed significant downregulation of CDK1, CDK2 and CDK4, and upregulation of the inhibitor CDKN1A, suggesting altered regulation of cell cycle progression. Collectively, these results show near complete compartmentalization of the fetal immune response to infected fetuses and suggest this immune response is not a major contributor to fetal death.

Maternal and fetal thyroid dysfunction following porcine reproductive and respiratory syndrome virus2 infection.

To better understand the host response to porcine reproductive and respiratory virus-2 (PRRSV2) we evaluated circulating thyroid hormone and associated gene expression in a late gestation challenge model. Pregnant gilts were inoculated at gestation day 85 and fetal samples collected at either 12 or 21 days post-infection (dpi). A subset of fetuses was selected for analysis based on viability and viral load categorized as either uninfected-viable (UNIF), high viral load viable (HV-VIA) or high viral load meconium stained (HV-MEC) and were compared with gestational age matched controls (CON). In dams, circulating levels of total T3 and T4 decreased in the acute period following infection and rebounded by 21 dpi. A similar effect was observed in fetuses, but was largely restricted to HV-VIA and HV-MEC, with minimal decrease noted in UNIF relative to CON at 21 dpi. Gene expression in fetal heart at 12 dpi showed significant decompensatory transcription of thyroid hormone transporters (SLC16A2) and deiodinases (DIO2, DIO3), which was not observed in brain. Correspondingly, genes associated with cell cycle progression (CDK1,2,4) were downregulated in only the heart of highly infected fetuses, while expression of their inhibitor (CDKN1A) was upregulated in both tissues. Finally, expression of genes associated with cardiac stress including CAMKD and AGT were upregulated in the hearts of highly infected fetuses, and a shift in expression of MYH6 to MYH7 was observed in HV-MEC fetuses specifically. Collectively, the results suggest PRRSV2 infection causes a hypothyroid state that disproportionally impacts the fetal heart over the brain.

Fermitin family homolog-2 (FERMT2) is highly expressed in human placental villi and modulates trophoblast invasion.

BACKGROUND: Integrins are transmembrane receptors that mediate cell-extracellular matrix (ECM) and cell-cell adhesion and trophoblast cells undergo changes in integrin expression as they differentiate. However, the mechanism(s) of integrin activation leading to integrin-mediated signaling in trophoblast cell differentiation is unknown. The Fermitin family proteins are integrin activators that help mediate integrin-mediated signaling, but have never been studied in detail within the human placenta. Thus, we examined the spatiotemporal pattern of expression of Fermitin family homolog-2 (FERMT2) in human chorionic villi throughout gestation and its role in trophoblast-substrate adhesion and invasion. METHODS: Placental villous tissue was obtained from patients undergoing elective terminations by dilatation and curettage at weeks 8-12 (n = 10), weeks 13-14 (n = 8), as well as from term deliveries at weeks 37-40 (n = 6). Tissues were fixed, processed and sections utilized for immunofluorescence analysis of FERMT2 expression during gestation. Additionally, HTR8-SVneo human trophoblast cells were transfected by electroporation with FERMT2-specific siRNAs or non-targeting siRNAs (control) and used in cell-substrate adhesion as well as invasion assays. RESULTS: FERMT2 was more commonly expressed in the basal domain of villous cytotrophoblast cells and prominently localized around the periphery of individual extravillous trophoblast cells. siRNA-mediated knockdown of FERMT2 in HTR8-SVneo cells resulted in significantly decreased trophoblast-substrate attachment (p < 0.05) as well as significantly decreased trophoblast invasion (p < 0.05) relative to control cells. CONCLUSIONS: The detection of FERMT2 throughout extravillous trophoblast columns and the results of invasion assays demonstrated that this protein is likely an important regulator of integrin activation in extravillous cells to modulate migration and invasion.

Insulinotropic nucleobindin-2/nesfatin-1 is dynamically expressed in the haemochorial mouse and human placenta.

The placenta is the physiological bridge between mother and fetus and has life-sustaining functions during pregnancy, including metabolic regulation, fetal protection and hormone secretion. Nucleobindin-2 (NUCB2) is a calcium- and DNA-binding protein and precursor of nesfatin-1, a signalling peptide with multiple functions, including regulation of energy homeostasis and glucose transport. These are also key functions of the placenta, yet NUCB2/nesfatin-1 expression has never been comprehensively studied in this organ. In the present study, mouse placental samples from Embryonic Day (E) 7.5 to E17.5 and human chorionic villi from the first and second trimester, as well as term pregnancy, were analysed for NUCB2/nesfatin-1 expression by immunohistochemistry with an antiserum that recognised both NUCB2 and nesfatin-1. From E7.5 to E9.5, NUCB2/nesfatin-1 was expressed in the ectoplacental cone, then parietal trophoblast giant cells and early spongiotrophoblast. At E10.5-12.5, NUCB2/nesfatin-1 expression became detectable in the developing labyrinth. From E12.5 and onwards, NUCB2/nesfatin-1 was expressed in the glycogen trophoblast cells, as well as highly expressed in syncytiotrophoblast, sinusoidal trophoblast giant cells and fetal capillary endothelial cells of the labyrinth. In all trimesters of human pregnancy, NUCB2/nesfatin-1 was highly expressed in syncytiotrophoblast. In addition, there was a significant increase in NUCB2 expression in human primary trophoblast cells induced to syncytialise. Thus, the haemochorial mammalian placenta is a novel source of NUCB2/nesfatin-1 and likely a site of its action, with potential roles in glucose homeostasis and/or nutrient sensing.

Histologic Changes Associated With Placental Separation in Gilts Infected with Porcine Reproductive and Respiratory Syndrome Virus.

The placenta is a vital organ providing the developing fetus with nutrient and gas exchange, thermoregulation, and waste elimination necessary for fetal development, as well as producing hormones to maintain pregnancy. It is hypothesized that fetal pig death in porcine reproductive and respiratory syndrome may be attributed to pathology of the maternal-fetal interface leading to premature placental separation. This study was designed to evaluate the chronologic progression of porcine reproductive and respiratory syndrome virus (PRRSV)-induced lesions at the maternal-fetal interface, with particular focus on placental separation in experimentally challenged third-trimester gilts. Fifteen gilts were inoculated with a virulent strain of PRRSV-2 on gestation day 86 ± 0.4. On multiple days postinoculation, 3 gilts along with 1 sham-inoculated control per time point were euthanized, and uterine and fetal placental tissues corresponding to each fetus were collected for histopathologic evaluation. The presence of any fetal lesion was 23 times more likely in compromised (meconium-stained and decomposed) compared with viable fetuses ( P < .001). In PRRSV-infected gilts, endometritis was more severe than placentitis, and the severity of endometrial inflammation and vasculitis increased progressively from 2 to 14 days postinoculation. Neither placental vasculitis nor a chronologic progression in the severity of placental detachment was observed. Severe placental detachment was more frequently present in PRRSV-infected compared with noninfected samples and was most significantly associated with placental inflammation, compared with other uterine lesions, viral load, or termination day. The results of this study suggest that placental separation by itself is not sufficient to significantly compromise fetal viability in reproductive porcine reproductive and respiratory syndrome.

Integrin linked kinase regulates syncytialization of BeWo trophoblast cells.

During placental development, mononuclear villous cytotrophoblast cells differentiate and fuse with the overlying syncytiotrophoblast. This process requires the dissolution of E-cadherin (CDH1)-containing adherens junctions in cytotrophoblast. Integrin linked kinase (ILK) can downregulate CDH1 through poly (ADP-ribose) polymerase 1 (PARP1) and Snail-1 (SNAI1) during epithelial-mesenchymal transition. ILK is known to be expressed in cytotrophoblast; thus, the role of a potential ILK-PARP1-SNAI1 pathway in aiding trophoblast syncytialization via the downregulation of CDH1 was examined. The spatiotemporal expression of PARP1, SNAI1, and CDH1 were determined in first and early second trimester chorionic villi, term villi, and BeWo cells by immunofluorescence analysis. PARP1 and SNAI1 were highly detectable in villous cytotrophoblast nuclei of human chorionic villi and SNAI1 expression, in particular, also persisted in syncytiotrophoblast. In BeWo cells undergoing syncytialization, PARP1 and SNAI1 increasingly localized to cell nuclei in correlation with decreased CDH1 expression. Using luciferase reporter assays, it was determined that PARP1 and SNAI1 promoter activities were significantly higher in BeWo cells during syncytialization compared to the activities in proliferating cells. Overexpression of wild type or constitutively active ILK also resulted in significantly increased PARP1 and SNAI1 promoter activities while dominant negative ILK overexpression significantly reduced promoter activities. Lastly, siRNA-mediated depletion of ILK expression in BeWo cells undergoing syncytialization resulted in significantly reduced SNAI1 expression and a significant reduction in the incidence of syncytialization correlating with increased CDH1 expression. These results demonstrate that ILK aids trophoblast syncytialization via the downregulation of CDH1, perhaps through an ILK-PARP1-SNAI1 pathway.

The Potential Functions of Small Heat Shock Proteins in the Uterine Musculature during Pregnancy.

The small heat shock protein B (HSPB) family is comprised of eleven members with many being induced by physiological stressors. In addition to being molecular chaperones, it is clear these proteins also play important roles in cell death regulation, cytoskeletal rearrangements, and immune system activation. These processes are important for the uterine smooth muscle or myometrium during pregnancy as it changes from a quiescent tissue, during the majority of pregnancy, to a powerful and contractile tissue at labor. The initiation and progression of labor within the myometrium also appears to require an inflammatory response as it is infiltrated by immune cells and it produces pro-inflammatory mediators. This chapter summarizes current knowledge on the expression of HSPB family members in the myometrium during pregnancy and speculates on the possible roles of these proteins during myometrial programming and transformation of the myometrium into a possible immune regulatory tissue.

Relationships of CD163 and CD169 positive cell numbers in the endometrium and fetal placenta with type 2 PRRSV RNA concentration in fetal thymus.

Several routes of porcine reproductive and respiratory virus PRRSV transmission across the porcine diffuse epitheliochorial placentation have been proposed, but none have been proven. The objectives of this study were to investigate associations between numbers of CD163 and CD169 positive macrophages, cathepsin positive areolae, and type 2 PRRSV load at the maternal-fetal interface in order to examine important factors related to transplacental infection. On gestation day 85 ± 1, naïve pregnant gilts were inoculated with PRRSV (n = 114) or were sham inoculated (n = 19). At 21 days post-inoculation (dpi), dams and their litters were humanely euthanized and necropsied. Samples of the maternal-fetal interface (uterus with fully attached placenta) and fetal thymus were collected for analysis by RT-qPCR to quantify PRRSV RNA concentration. The corresponding paraffin-embedded uterine tissue sections were subjected to immunohistochemistry for PRRSV nucleocapsid N protein, CD163, CD169, and cathepsin. Our findings confirm significant increases in the numbers of PRRSV, CD163 and CD169 positive cells at the maternal-fetal interface during type 2 PRRSV infection in pregnant gilts. PRRSV load in fetal thymus was positively related to CD163(+) cell count in endometrium and negatively related to CD163(+) cell count in placenta, but unrelated to CD169 counts or cathepsin positive areolae. The endometrium:placenta ratio of CD163 cells, and to a lesser extent CD169 cells, was significantly associated with an increase fetal viral load in thymus. These findings suggest a more important role for CD163(+) cells following trans-placental PRRSV infection, but dichotomous responses in endometrium and placenta for both CD163 and CD169 cells.

HSPB8 and the Cochaperone BAG3 Are Highly Expressed During the Synthetic Phase of Rat Myometrium Programming During Pregnancy.

The small heat shock protein (HSP) B family of proteins are a group of molecular chaperones that enable tissues to adapt to changes in their local environments during differentiation, stress, or disease conditions. The objective of this research was to characterize the expression of HSPB8 and its cochaperone Bcl2-associated athanogene 3 (BAG3) in nonpregnant (NP) and pregnant rat myometrium during myometrial programming. Rat myometrium was collected from NP and pregnant rats as well as 1 day postpartum (PP) and samples prepared for immunoblot and immunofluorescence analysis. Immunoblot analysis determined that HSPB8 protein expression was significantly elevated at Day (D) 15, D17, and D19 compared to expression at NP and D6, while BAG3 expression was significantly elevated at D15 compared to NP, and D17 compared to NP, D6, D23, and PP time points (P < 0.05). In situ, HSPB8 and BAG3 were predominantly localized to myometrial cells throughout pregnancy, with intense cytoplasmic HSPB8 and BAG3 detection on D15 and D17 in both longitudinal and circular muscle layers. Immunoblot analysis of HSPB8 and BAG3 protein expression in myometrium from unilateral pregnancies also revealed that expression of both proteins was significantly increased at D15 in gravid compared to nongravid horns. Thus, HSPB8 and BAG3 are highly expressed during the synthetic phase of myometrial differentiation marked by initiation of uterine distension and myometrial hypertrophy. HSPB8 and BAG3 could be regulators of the protein quality control required for this process.

Effect of porcine reproductive and respiratory syndrome virus 2 on tight junction gene expression at the maternal-fetal interface.

Understanding why intrauterine growth restricted (IUGR) fetuses are more resilient to transplacental porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) infection compared to normal fetuses may lead to alternative approaches to control PRRS. Our objective was to compare gene expression of a subset of tight junction proteins in the endometrium (END) and placenta (PLC) of i) IUGR vs N-IUGR fetuses, and ii) across disease progression phenotypes following PRRSV-2 infection. In experiment 1, snap frozen END and PLC from fetuses of non-infected control dams (CTRL) and from high viral load viable (HVL-VIA) fetuses, with both groups further classified as either IUGR or non(N)-IUGR based on brain: liver weight ratio were strategically selected from a large challenge trial. In experiment 2, similar tissues were randomly selected from CTRL and from uninfected thymus (UNIF), (HVL-VIA) and HVL meconium-stained in the body (HVL-MEC-B) of PRRSV-infected dams. The expression of claudin (CLDN) 1, 3, 4, 5, 6, 7, 10, tight junction protein 1 (TJP1) and occludin (OCLN) genes were evaluated by PCR. There were no significant group differences between IUGR and N-IUGR groups, regardless of infection status, that explained the resilience of IUGR fetuses. Regarding disease progression, elevated CLDN3 was observed in END of UNIF, CLDN6 expression was lower in PLC when the fetus became infected (HVL-VIA), and CLDN10 elevated in PLC in fetuses showing evidence of compromise (HVL-MEC-B). Lastly, OCLN gene expression was higher in the END and PLC following maternal infection. In conclusion, differences in TJ integrity were mainly observed following PRRSV-2 infection with stepwise changes corresponding with disease progression.

Detection of PRRSV-2 alone and co-localized with CD163 positive macrophages in porcine placental areolae.

The porcine epitheliochorial placenta creates a barrier for the transplacental transfer of some nutrients from the dam to the fetus, as well as feto-lethal viruses such as porcine reproductive and respiratory syndrome virus 2 (PRRSV-2). Areolae are specialized structures within the porcine placenta with a high absorptive and substance transport capacity that facilitate embryonic development. The overarching aim of this study was to characterize the localization of PRRSV-2 in and adjacent to areolae to provide insight into whether transplacental transmission might occur through placental areolae. Control (CON) plus three phenotypic fetal groups were selected based on levels of virus in fetal placenta, sera and thymus, to determine if fetal resilience was related to differences in PRRSV-2 localization, alone or co-localized with CD163+ macrophages. These fetal groups represented a range of susceptibility: uninfected (UNINF) being resistant, infected in placenta only (PLCO) being resilient, and high viral load viable (HVL-VIA) being most susceptible. Finally, potential factors related to PRRSV-2 localization, including the severity of inflammation in endometrium and placenta, and intrauterine growth restriction, known resilience factors, were assessed. Thirty-one pregnant gilts were inoculated with PRRSV-2 at gestation day 86 ± 0.4. Seven pregnant gilts were sham-inoculated. Gilts were euthanized at 12 days post-infection. Presence of PRRSV and CD163+ macrophages were determined using immunofluorescence in cryosections of maternal-fetal interface (MFI) with and without areolae. In the maternal, fetal and cavity of areolar region PRRSV particles were found both independently and co-localized with CD163+ macrophages. Similarly, individual, and co-localized particles were observed in the maternal and fetal sides of the MFI region of infected fetuses. Weak positive correlations were observed between the counts of CD163+ macrophages and some inflammation scores in endometrial and placental tissues, but no correlations with PRRSV-2 localization. There were no differences across the four fetal groups evaluated. These results suggest that transplacental transmission of PRRSV may occur through the areolae, either as non-cell associated or in association with infected CD163 macrophages.

Decreased tight junction protein intensity in the placenta of porcine reproductive and respiratory syndrome virus-2 infected fetuses.

INTRODUCTION: Existing strategies to control porcine reproductive and respiratory syndrome (PRRS) are not completely effective and require alternative approaches. Although intrauterine growth restricted (IUGR) fetuses are more resilient to transplacental PRRS virus-2 (PRRSV2) infection compared to normal fetuses, the exact mechanisms are unknown. The objective of this research was to assess abundance and localization of a subset of tight junction (TJ) proteins in the maternal-fetal interface and any alterations that may affect the movement of nutrients or PRRSV2 across the epitheliochorial placenta. METHODS: Paraffin-embedded samples of placenta from non-infected control (CTRL) and PRRSV2 infected fetuses (IUGR, non(N)-IUGR, meconium-stained (MEC) (n = 6 per group) were randomly selected from a large challenge trial and immunostained for claudins (CLDN) 1, 3, 4, 7 and tight junction protein 1 (TJP1). Immunostaining intensity was semi-subjectively scored by region. RESULTS: Intensity of CLDN1 was lower in placenta of IUGR, MEC, and N-IUGR fetuses compared to CTRL, mainly in fetal epithelium and maternal endothelial cells (MECL). CLDN4 intensity was lower in MECL of IUGR compared to CTRL and MEC fetuses. TJP1 intensity was lower in maternal and fetal epithelia of placenta within IUGR, MEC, and N-IUGR fetuses versus CTRL. DISCUSSION: Differences were mainly observed between PRRSV2 infected and non-infected groups indicating TJ integrity was affected by PRRSV2 infection. These results provide insights into the potential mechanisms of transplacental transmission of PRRSV2; however, since only CLDN4 differed amongst the infected groups, PRRSV2 induced changes in TJ integrity do not appear to explain variation in fetal outcomes after infection.

Macroscopic, Histologic, and Immunomodulatory Response of Limb Wounds Following Intravenous Allogeneic Cord Blood-Derived Multipotent Mesenchymal Stromal Cell Therapy in Horses.

Limb wounds are common in horses and often develop complications. Intravenous multipotent mesenchymal stromal cell (MSC) therapy is promising but has risks associated with intravenous administration and unknown potential to improve cutaneous wound healing. The objectives were to determine the clinical safety of administering large numbers of allogeneic cord blood-derived MSCs intravenously, and if therapy causes clinically adverse reactions, accelerates wound closure, improves histologic healing, and alters mRNA expression of common wound cytokines. Wounds were created on the metacarpus of 12 horses. Treatment horses were administered 1.51-2.46 × 108 cells suspended in 50% HypoThermosol FRS, and control horses were administered 50% HypoThermosol FRS alone. Epithelialization, contraction, and wound closure rates were determined using planimetric analysis. Wounds were biopsied and evaluated for histologic healing characteristics and cytokine mRNA expression. Days until wound closure was also determined. The results indicate that 3/6 of treatment horses and 1/6 of control horses experienced minor transient reactions. Treatment did not accelerate wound closure or improve histologic healing. Treatment decreased wound size and decreased all measured cytokines except transforming growth factor-ß3. MSC intravenous therapy has the potential to decrease limb wound size; however, further work is needed to understand the clinical relevance of adverse reactions.

Samples sizes required to accurately quantify viral load and histologic lesion severity at the maternal-fetal interface of PRRSV-inoculated pregnant gilts.

Porcine reproductive and respiratory syndrome virus (PRRSV) is transmitted vertically, causing fetal death in late gestation. Spatiotemporal distribution of virus at the maternal-fetal interface (MFI) is variable, and accurate assessment of viral concentration and lesions is thus subject to sampling error. Our objectives were: 1) to assess whether viral load and lesion severity in a single sample of endometrium (END) and placenta (PLC), collected near the base of the umbilical cord (the current standard), are representative of the entire organ; and 2) to compare sampling strategies and evaluate if spatial variation in viral load can be overcome by pooling of like-tissues. Spatially distinct pieces of END and PLC of 24 fetuses from PRRSV-2-infected dams were collected. PRRSV RNA quantified by RT-qPCR was compared in 5 individual pieces per fetus and in respective pools of tissue and extracted RNA. Three distinct pieces of MFI were assessed for histologic severity. Concordance correlation and kappa inter-rater agreement were used to characterize agreement among individual samples and pools. The viral load of individual samples and pools of END had greater concordance to a referent standard than did samples of PLC. Larger pool sizes had greater concordance than smaller pool sizes. Average viral load and lesion severity did not differ by location sampled, and no technical advantages of pooling tissues versus RNA extracts were found. We conclude that multiple pieces of MFI tissues must be evaluated to accurately assess lesion severity and viral load. Three pieces per fetus provided a reasonable balance of cost and logistic feasibility.

Thyroid hormone suppression in feeder pigs following polymicrobial or porcine reproductive and respiratory syndrome virus-2 challenge.

Thyroid hormones are powerful regulators of growth, development, and basal metabolic rate and can be dysregulated under conditions of severe stress or illness. To understand the role of these hormones in porcine disease response, serum samples were obtained from three batches of nursery-aged pigs (n = 208) exposed to a natural polymicrobial disease challenge with an array of bacterial and viral pathogens. Levels of total thyroxin (T4) and triiodothyronine (T3) assessed in sera by radioimmunoassay, decreased significantly by 14 days post-exposure (DPE). Levels of T3 partially rebounded by 48 DPE, while T4 levels remain depressed. Post-exposure T3 and T4 levels were positively correlated with acute and long-term average daily gain (ADG). Cross-sectional sampling of animals maintained at the high health source farms, showed no equivalent change in either hormone when managed under standard industrial conditions. To further elucidate the effect of porcine reproductive and respiratory syndrome virus (PRRSV)-infection on thyroid hormone levels, archived sera over 42 days post inoculation (DPI) from nursery pigs (N = 190) challenged with one of two PRRSV2 strains by the PRRS Host Genetics Consortium were similarly assessed, with animals selected in a two-by-two design, to investigate biological extremes in ADG and viral load (VL). All animals showed a similar decrease in both thyroid hormones reaching a minimum at 7 DPI and returning to near pre-challenge levels by 42 DPI. Post-challenge T3 and T4 levels were significantly greater in high ADG groups, with no significant association with VL or strain. The results of this study demonstrate porcine susceptibility to thyroid disruption in response to disease challenge and demonstrate a relationship between this response and growth performance.