Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection of pregnant pigs can result in congenital infection and ultimately fetal death. Little is known about immune responses to ...infection at the maternal-fetal interface and in the fetus itself, or the molecular events behind virus transmission and disease progression in the fetus. To investigate these processes, RNA-sequencing of two sites, uterine endothelium with adherent placental tissue and fetal thymus, was performed 21 days post-challenge on four groups of fetuses selected from a large PRRSV challenge experiment of pregnant gilts: control (CON), uninfected (UNINF), infected (INF), and meconium-stained (MEC) (n = 12/group). Transcriptional analyses consisted of multiple contrasts between groups using two approaches: differential gene expression analysis and weighted gene co-expression network analysis (WGCNA). Biological functions, pathways, and regulators enriched for differentially expressed genes or module members were identified through functional annotation analyses. Expression data were validated by reverse transcription quantitative polymerase chain reaction (RTqPCR) carried out for 16 genes of interest.
The immune response to infection in endometrium was mainly adaptive in nature, with the most upregulated genes functioning in either humoral or cell-mediated immunity. In contrast, the expression profile of infected fetal thymus revealed a predominantly innate immune response to infection, featuring the upregulation of genes regulated by type I interferon and pro-inflammatory cytokines. Fetal infection was associated with an increase in viral load coupled with a reduction in T cell signaling in the endometrium that could be due to PRRSV-controlled apoptosis of uninfected bystander cells. There was also evidence for a reduction in TWIST1 activity, a transcription factor involved in placental implantation and maturation, which could facilitate virus transmission or fetal pathology through dysregulation of placental function. Finally, results suggested that events within the fetus could also drive fetal pathology. Thymus samples of meconium-stained fetuses exhibited an increase in the expression of pro-inflammatory cytokine and granulocyte genes previously implicated in swine infectious disease pathology.
This study identified major differences in the response to PRRSV infection in the uterine endometrium and fetus at the gene expression level, and provides insight into the molecular basis of virus transmission and disease progression.
Minimal research has focused on understanding mechanisms underlying porcine reproductive and respiratory syndrome virus (PRRSV) induced reproductive failure. We have completed a large-scale project ...investigating phenotypic and genotypic predictors of reproductive PRRS severity in which numerous clinical, pathological, immunologic and viral responses were characterized in dams and fetuses. The goal was to determine which phenotypic responses were associated with fetal viral load and death after experimental infection of pregnant gilts with type 2 PRRSV, thereby elucidating mechanisms of reproductive PRRS in third trimester pregnant gilts. The presence of fetal infection and increasing RNA concentration at the maternal-fetal interface were strong predictors of the probability of fetal death, while PRRSV RNA concentration in dam sera and systemic tissues were not associated with the odds of fetal death. Fetal infection and death clustered, indicating that the status of adjacent fetuses is crucial for lateral transmission and fetal outcome. Several systemic immune responses of gilts were associated with fetal outcome and viral load: interferon-α contributed to the probability of fetal death, but absolute numbers of T helper cells in early infection, absolute numbers of myeloid cells over time and interleukin 12 levels appeared protective. These results suggest specific immune responses may either contribute to, or protect against, transplacental virus transmission. The WUR10000125 SNP on chromosome 4, associated with PRRS resilience in nursery pigs, was not associated with reproductive outcome. Whereas past research suggested that fetal death results from events occurring at the maternal-fetal interface, we conclude that viral replication within fetuses and spread of PRRSV to adjacent fetuses are pivotal events in the pathogenesis of reproductive PRRS.
•ETN is suggested as an infectious disease, not related to viruses.•Intradermal inoculation triggers the initial lesions characteristic of ETN and resulted in partial loss of pinnae cartilage.•The ...full aetiology, particularly related to progression, remains unclear.
Swine ear-tip necrosis (ETN) is a disease of global presence and unclear aetiology. Little evidence is available regarding the nature of this disease. The aim of this work was to investigate if ETN is an infectious disease that could be replicated using a lesion macerate inoculum.
A source farm with a history of ear-tip necrosis was identified and five weeks-old pigs (n = 12) from this farm were housed under controlled conditions and intradermally inoculated with ETN lesion macerates (right ear, n = 10) or sterile inoculum (left ear, n = 10). Two pigs were not inoculated, serving as sentinels. All animals were clinically monitored daily during 21 days, and a ETN ear score was used to follow disease progression. Anaerobic (n = 2) and aerobic (n = 2) overnight cultures, as well as raw aliquots of the lesion macerate inoculum (n = 2) and control inoculum (n = 2) were submitted for metagenomic sequencing. All inoculated ears developed lesions suggestive of early ETN, but none progressed to result in loss of the ear pinna. All completely resolved 21 days post-inoculation. Post-mortem investigation revealed areas of fibrosis, characterized by a granulomatous response in the inoculated ears (5/10) and in 1/10 control ears. Metagenomic analysis identified the presence of previously suggested bacterial etiological agents, but no relevant viral, fungal or protozoan agents in the inoculum. ETN etiology remains unclear, but an infectious cause and bacterial agents are suggested to be at least partially implicated in disease pathogenesis. Virus and fungi do not seem to significantly contribute to disease.
The objective of this study was to extract novel phenotypes related to disease resilience using daily feed intake data from growing pigs under a multifactorial natural disease challenge that was ...designed to mimic a commercial environment with high disease pressure to maximize expression of resilience. Data used were the first 1,341 crossbred wean-to-finish pigs from a research facility in Québec, Canada. The natural challenge was established under careful veterinary oversight by seeding the facility with diseased pigs from local health-challenged farms, targeting various viral and bacterial diseases, and maintaining disease pressure by entering batches of 60-75 pigs in a continuous flow system. Feed intake (FI) is sensitive to disease, as pigs tend to eat less when they become ill. Four phenotypes were extracted from the individual daily FI data during finishing as novel measures of resilience. The first two were daily variability in FI or FI duration, quantified by the root mean square error (RMSE) from the within individual regressions of FI or duration at the feeder (DUR) on age (RMSE
and RMSE
). The other two were the proportion of off-feed days, classified based on negative residuals from a 5% quantile regression (QR) of daily feed intake or duration data on age across all pigs (QR
and QR
). Mortality and treatment rate had a heritability of 0.13 (±0.05) and 0.29 (±0.07), respectively. Heritability estimates for RMSE
, RMSE
, QR
, and QR
were 0.21 (±0.07) 0.26 (±0.07), 0.15 (±0.06), and 0.23 (±0.07), respectively. Genetic correlations of RMSE and QR measures with mortality and treatment rate ranged from 0.37 to 0.85, with QR measures having stronger correlations with both. Estimates of genetic correlations of RMSE measures with production traits were typically low, but often favorable (e.g., -0.31 between RMSE
and finishing ADG). Although disease resilience was our target, fluctuations in FI and duration can be caused by many factors other than disease and should be viewed as overall indicators of general resilience to a variety of stressors. In conclusion, daily variation in FI or duration at the feeder can be used as heritable measures of resilience.
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.
•PRRS resilience in IUGR fetuses was not related to tight junction expression.•Tight junction expression was modified in diseased progression groups.•OCLN expression increased in endometrium and placenta after maternal infection.•CLDN6 expression decreased in placenta when the fetus became infected.•CLDN10 expression increased in placenta in fetuses showing evidence of compromise.
Selection for disease resilience, which refers to the ability of an animal to maintain performance when exposed to disease, can reduce the impact of infectious diseases. However, direct selection for ...disease resilience is challenging because nucleus herds must maintain a high health status. A possible solution is indirect selection of indicators of disease resilience. To search for such indicators, we conducted phenotypic and genetic quantitative analyses of the abundances of 377 proteins in plasma samples from 912 young and visually healthy pigs and their relationships with performance and subsequent disease resilience after natural exposure to a polymicrobial disease challenge. Abundances of 100 proteins were significantly heritable (false discovery rate (FDR) <0.10). The abundance of some proteins was or tended to be genetically correlated (rg) with disease resilience, including complement system proteins (rg = -0.24, FDR = 0.001) and IgG heavy chain proteins (rg = -0.68, FDR = 0.22). Gene set enrichment analyses (FDR < 0.2) based on phenotypic and genetic associations of protein abundances with subsequent disease resilience revealed many pathways related to the immune system that were unfavorably associated with subsequent disease resilience, especially the innate immune system. It was not possible to determine whether the observed levels of these proteins reflected baseline levels in these young and visually healthy pigs or were the result of a response to environmental disturbances that the pigs were exposed to before sample collection. Nevertheless, results show that, under these conditions, the abundance of proteins in some immune-related pathways can be used as phenotypic and genetic predictors of disease resilience and have the potential for use in pig breeding and management.
Abstract
Background
The pork industry faces unprecedented challenges from disease, which increases cost of production and use of antibiotics, and reduces production efficiency, carcass quality, and ...animal wellbeing. One solution is to improve the overall resilience of pigs to a broad array of common diseases through genetic selection. Behavioral changes in feeding and drinking are usually the very first clinical signs when animals are exposed to stressors such as disease. Changes in feeding and drinking behaviors in diseased pigs may reflect the way they cope with the challenge and, thus, could be used as indicator traits to select for disease resilience. The objectives of this study were to estimate genetic parameters of feeding and drinking traits for wean-to-finish pigs in a natural polymicrobial disease challenge model, to estimate genetic correlations of feeding and drinking traits with growth rate and clinical disease traits, and to develop indicator traits to select for disease resilience.
Results
In general, drinking traits had moderate to high estimates of heritability, especially average daily water dispensed, duration, and number of visits (0.44 to 0.58). Similar estimates were observed for corresponding feeding traits (0.35 to 0.51). Most genetic correlation estimates among drinking traits were moderate to high (0.30 to 0.92) and higher than among feeding traits (0 to 0.11). Compared to other drinking traits, water intake duration and number of visits had relatively stronger negative genetic correlation estimates with treatment rate and mortality, especially across the challenge nursery and finisher (− 0.39 and − 0.45 for treatment rate; − 0.20 and − 0.19 for mortality).
Conclusion
Most of the recorded drinking and feeding traits under a severe disease challenge had moderate to high estimates of heritability, especially for feed or water intake duration and number of visits. Phenotypic and genetic correlations among the recorded feeding traits under disease were generally low but drinking traits showed high correlations with each other. Water intake duration and number of visits are potential indicator traits to select for disease resilience because of their high heritability and had moderate genetic correlations with treatment and mortality rates under severe disease.
•Existing data from multiple divergent sources can be useful in monitoring temporal trends.•Clinical syndromic surveillance should be coupled with a good intelligence network to interpret and ...disseminate the results.•Clinical impression survey data can be used in stochastic scenario tree models for disease freedom.
The Canada West Swine Health Intelligence Network (CWSHIN) is a surveillance system imbedded in an intelligence network. It has been conducting syndromic surveillance in the four western provinces of Canada since 2012. The quarterly activities include repeated clinical impression surveys, compilation of laboratory data, discussion of trends with an expert group (practitioners, laboratory diagnosticians) and swine health reports for producers and swine practitioners. However, due to the lack of standardized population identifiers across data sources usual methods of combining data could not be applied and the collated data were not being fully utilized and analysed. Therefore in 2019, CWSHIN underwent a substantial review resulting in the “Next Generation CWSHIN”.
The objectives of this study were to develop and evaluate a new data merging method to combine CWSHIN’s clinical impression survey and laboratory data; and to provide examples of analyses and modeling based on these data.
The data for analysis were restricted to repeated clinical impression surveys (2019–2020) from veterinary practitioners and laboratory diagnostic data (2016–2020). Merging surveillance data from existing sources can be challenging. Therefore, as an alternative to merge data using a hierarchy of population identifiers, we developed a Disease Map to link surveillance data from all our data-sources. The resulting Data Repository allowed monitoring of temporal trends of syndromes, clinical diseases, and laboratory identified organisms, but it cannot provide estimates of disease occurrence. Two main reasons were the lack of denominators and using existing data on routine diagnostic tests. Therefore, discussion in the expert group (veterinary practitioners, laboratory diagnosticians, swine health experts) was critical to the system’s success. Based on repeated clinical impression surveys a stochastic scenario tree model for freedom from Foot and Mouth Disease (CWSHIN Blister model) was also developed.
In conclusion, the method to link existing data systems from multiple divergent sources by means of a Disease Map improved CWSHIN’s veterinary syndromic surveillance. Together the Data Repository and Disease map provided flexibility to monitor temporal trends, define populations and diseases, and allow analysis. However, it is critical that the surveillance is coupled with a good intelligence network that can help interpret the results and disseminate knowledge to veterinarians and producers.
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.
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.
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.
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.
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.
•A subset of tight junctions is detected in all regions of the porcine placenta.•PRRSV2 reduced tight junction intensity in epithelium of the porcine placenta.•PRRSV2 reduced tight junction intensity in endothelium of the porcine placenta.•Claudin-4 intensity decreased in endothelial cells of meconium-stained fetuses.•Altered tight junction intensity does not explain PRSV2 resilience of IUGR fetuses.
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