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.

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.

Evaluation of inhibition of F4ac positive Escherichia coli attachment with xanthine dehydrogenase, butyrophilin, lactadherin and fatty acid binding protein.

BACKGROUND: Neonatal and post-weaning colibacillosis caused by enterotoxigenic E. coli is responsible for substantial economic losses encountered by the pork industry. Intestinal colonization of young piglets by E. coli depends on the efficiency of bacterial attachment to host gastrointestinal epithelium that is mediated by fimbriae. We tested the effect of porcine individual milk fat globule membrane (MFGM) proteins on F4ac positive E. coli attachment to porcine enterocytes in vitro. RESULTS: Butyrophilin, lactadherin and fatty acid binding protein inhibited fimbriae-dependent adherence of E. coli to enterocytes in vitro, while xanthine dehydrogenase did not. The inhibiting activity was dose-dependent for all three proteins, but the inhibiting efficiency was different. CONCLUSIONS: The results indicate that MFGM proteins may interfere with attachment of E. coli to porcine neonatal intestinal mucosa.

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.

Classification of fetal resilience to porcine reproductive and respiratory syndrome (PRRS) based on temporal viral load in late gestation maternal tissues and fetuses.

Although porcine reproductive and respiratory syndrome virus (PRRSV) readily crosses the maternal fetal interface (MFI) in third trimester, fetal resilience varies within litters. The aim of this study was to characterize PRRSV-2 concentration in MFI and fetuses at five time points after experimental inoculation of late gestation gilts and use this information to classify potentially resistant, resilient and susceptible fetuses. The secondary objective was to verify the relationship between PRRS viral load and intrauterine growth retardation (IUGR). Three PRRSV-inoculated pregnant gilts and 1 sham-inoculated control were euthanized at five time points in days post infection (DPI; 2, 5, 8, 12, 14). The preservation status of each fetus was determined and MFI samples adjacent to the umbilical stump of each fetus, as well as serum, thymus, umbilical cord and amniotic fluid were collected. Viral load was quantified using probe-based reverse-transcriptase quantitative PCR (RT-qPCR) targeting PRRSV NVSL 97-7895 ORF7. Our result show the MFI was largely PRRSV infected by 2 DPI and virus was first detected in fetal sera and umbilical cord by 5 DPI, and in fetal thymus and amniotic fluid by 8 DPI. This indicates that PRRSV-2 quickly crossed the placenta and traveled toward the fetus via umbilical circulation within one week of the dam's inoculation. Fetal compromise was first observed on 8 DPI and increased progressively through to 14 DPI. However, several factors were associated with fetal resilience. The random forest model identified that 'viral load in fetal thymus' and duration of infection ('DPI') as the most important factors predicting fetal resilience and resistance. Moreover, IUGR fetuses had lower viral load and were less frequently compromised or dead compared to non-IUGR and average cohorts. Understanding the mechanisms of fetal resilience to PRRSV will improve selection strategies for replacement gilts.

Type 2 porcine reproductive and respiratory syndrome virus infection increases apoptosis at the maternal-fetal interface in late gestation pregnant gilts.

The pathogenesis of fetal death associated with porcine reproductive and respiratory syndrome (PRRS) is hypothesized to be a consequence of PRRS virus-induced apoptosis at the maternal-fetal interface (MFI). The objectives of this study were to evaluate distribution and degree of apoptosis in the uterine and fetal placental tissues during the experimental type 2 PRRS virus (PRRSV) infection and determine associations between apoptosis at the MFI, PRRSV RNA concentration and antigen staining intensity, PRRSV-induced microscopic lesions, and fetal preservation status. A total of 114 naïve, high-health pregnant gilts were inoculated with type 2 PRRSV on gestation day 85±1 with euthanasia 21 days later; 19 sham-inoculated gilts served as controls. Two hundred and fifty samples of uterine tissue with fetal placenta were selected based on negative, low PRRSV RNA, and high PRRSV RNA concentration (0, < or > 2.7 log10 copies/mg, respectively). TUNEL assay was used to detect apoptosis in the endometrium and at the MFI. PRRSV RNA concentration and numbers of PRRSV immunopositive cells in uterine and placental tissue were positively associated with the severity of apoptosis in the endometrium and the MFI (P<0.001, P<0.05 and P<0.001, respectively). The number of TUNEL positive cells at the MFI was also positively associated with the severity (P<0.001) of vasculitis, but not total numbers of inflammatory cells in the endometrium. Increased numbers of TUNEL positive cells at the MFI were associated with PRRSV load in the fetal thymus, and greater odds of meconium staining of the fetus at 21 days post infection (P<0.001 for both). These findings suggest an important role of apoptosis in the pathogenesis of uterine epithelial and trophoblastic cell death at the MFI. Moreover, apoptosis at the MFI is significantly associated with fetal demise during in utero type 2 PRRSV infection.

Novel insights into host responses and reproductive pathophysiology of porcine reproductive and respiratory syndrome caused by PRRSV-2.

A large challenge experiment using North American porcine reproductive and respiratory virus (PRRSV-2) provided new insights into the pathophysiology of reproductive PRRS. Deep phenotyping of dams and fetuses identified maternal and fetal predictors of PRRS severity and resilience. PRRSV infection resulted in dramatic decreases in all leukocyte subsets by 2days post inoculation. Apoptosis in the interface region was positively related to endometrial vasculitis, viral load in endometrium and fetal thymus, and odds of meconium staining. Viral load at the maternal-fetal interface was a strong predictor of viral load in fetal thymus and odds of fetal death. However, interferon-alpha suppression, a consequence of PRRSV infection, was protective against fetal death. Although the prevalence of fetal lesions was low, their presence in fetal organs and umbilical cord was strongly associated with fetal compromise. Fetal death and viral load clustered in litters suggesting inter-fetal transmission starting from a limited number of index fetuses. Factors associated with index fetal infection are unclear, but large fetuses appear at greater risk. Disease progression in fetuses was associated with an up-regulation of genes associated with inflammation, innate immunity, and cell death signaling, and down-regulation of genes associated with cell cycle and lymphocyte quality. A number of maternal transcriptomic responses were associated with PRRS resilience including higher basal gene expression correlated with platelet function, interferon and pro-inflammatory responses. Twenty-one genomic regions across 10 chromosomes were associated with important traits including fetal viral load, fetal death and viability suggesting that selection for reproductive PRRS resilience may be possible.

Pathologic Evaluation of Type 2 Porcine Reproductive and Respiratory Syndrome Virus Infection at the Maternal-Fetal Interface of Late Gestation Pregnant Gilts.

The pathogenesis of fetal death caused by porcine reproductive and respiratory syndrome virus (PRRSV) remains unclear. The objective of this study was to improve our understanding of the pathogenesis by assessing potential relationships between specific histopathological lesions and PRRSV RNA concentration in the fetuses and the maternal-fetal interface. Pregnant gilts were inoculated with PRRSV (n = 114) or sham inoculated (n = 19) at 85±1 days of gestation. Dams and their litters were humanely euthanized and necropsied 21 days later. PRRSV RNA concentration was measured by qRT-PCR in the maternal-fetal interface and fetal thymus (n = 1391). Presence of fetal lesions was positively related to PRRSV RNA concentration in the maternal-fetal interface and fetal thymus (P<0.05 for both), but not to the distribution or severity of vasculitis, or the severity of endometrial inflammation. The presence of fetal and umbilical lesions was associated with greater odds of meconium staining (P<0.05 for both). The distribution and severity of vasculitis in endometrium were not significantly related to PRRSV RNA concentration in maternal-fetal interface or fetal thymus. Endometrial inflammation severity was positively related to distribution and severity of vasculitis in endometrium (P<0.001 for both). Conclusions from this study suggest that type 2 PRRSV infection in pregnant gilts induces significant histopathological lesions at maternal-fetal interface, but they are not associated with presence of PRRSV in the maternal-fetal interface at 21 days post infection. Conversely, fetal pathological lesions are associated with presence of PRRSV in the maternal-fetal interface and fetal thymus, and meconium staining is significantly associated with the presence of both fetal and umbilical lesions observed 21 days post infection.

Identification of Escherichia coli F4ac-binding proteins in porcine milk fat globule membrane.

F4ac-positive enterotoxigenic Escherichia coli (ETEC) must attach to the intestinal mucosa to cause diarrhea in piglets. Prevention of bacterial attachment to the intestinal mucosa is the most effective defense against ETEC-induced diarrhea. Porcine milk fat globule membranes (MFGM) were shown to be able to inhibit attachment of ETEC to the intestinal brush border; however, the specific components of porcine MFGM that inhibited attachment of ETEC to enterocytes were not identified. Accordingly, the purpose of this study was to identify F4ac-binding MFGM proteins by overlay Western blot and affinity chromatography. The proteome of porcine MFGM was characterized and the following F4ac-binding proteins were detected by overlay Western blot and affinity chromatography: lactadherin, butyrophilin, adipophilin, acyl-CoA synthetase 3, and fatty acid-binding protein 3. The biological function of these proteins was not investigated but it is possible that their interaction with F4ac fimbria interferes with bacterial attachment and colonization.

Les Escherichia coli entérotoxinogénique (ETEC) positif pour F4ac doivent s'attacher à la muqueuse intestinale pour causer la diarrhée chez les porcelets. L'empêchement de l'attachement bactérien à la muqueuse intestinale est le moyen de défense le plus efficace contre la diarrhée induite par les ETEC. Les membranes de globules de gras de lait porcin (MFGM) ont été montré comme étant capable d'inhiber l'attachement des ETEC à la bordure en brosse intestinale; toutefois, les composantes spécifiques des MFGM porcines qui inhibaient l'attachement des ETEC aux entérocytes ne furent pas identifiées. Ainsi, le but de la présente étude était d'identifier les protéines des MFGM liant F4ac par immunobuvardage par superposition et chromatographie d'affinité. Le protéome des MFGM porcine fut caractérisé et les protéines liant F4as suivantes furent détectées par immunobuvardage par superposition et chromatographie d'affinité : lactadhérine, butyrophiline, adipophiline, acyl-CoA synthétase 3, et la protéine 3 liant les acides gras. La fonction biologique de ces protéines ne fut pas étudiée mais il est possible que leur interaction avec les fimbriae F4ac interfère avec l'attachement bactérien et la colonisation.(Traduit par Docteur Serge Messier).