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Borrelia burgdorferi is a tick-borne spirochete that causes Lyme disease in humans. The host immune system controls the abundance of the spirochete in the host tissues. Recent work with immunocompetent Mus musculus mice strain C3H/HeJ found that males had a higher tissue infection prevalence and spirochete load compared to females. The purpose of this study was to determine whether host sex and acquired immunity interact to influence the prevalence and abundance of spirochetes in the tissues of the commonly used mouse strain C57BL/6. Wildtype (WT) mice and their SCID counterparts (C57BL/6) were experimentally infected with B. burgdorferi via tick bite. Ear biopsies were sampled at weeks 4, 8, and 12 post-infection (PI) and five tissues (left ear, ventral skin, heart, tibiotarsal joint of left hind leg, and liver) were collected at necropsy (16 weeks PI). The mean spirochete load in the tissues of the SCID mice was 260.4x higher compared to the WT mice. In WT mice, the infection prevalence in the ventral skin was significantly higher in males (40.0 %) compared to females (0.0 %), and the spirochete load in the rear tibiotarsal joint was significantly higher (4.3x) in males compared to females. In SCID mice, the spirochete load in the ventral skin was 200.0x higher in males compared to females, but there were no significant sex-specific difference in spirochete load in the other tissues (left ear, heart, tibiotarsal joint, or liver). Thus, the absence of acquired immunity greatly amplified the spirochete load in the ventral skin of male mice. It is important to note that the observed sex-specific differences in laboratory mice cannot be extrapolated to humans. Future studies should investigate the mechanisms underlying the male bias in the abundance of B. burgdorferi in the mouse skin., Competing Interests: Declaration of competing interests The authors declare that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier GmbH.)

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.

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

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.

Introduction: Porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) breaches the maternal-fetal interface (MFI) to infect porcine fetuses, yet the exact mechanism(s) of transmission is not understood. The objective of this study was to determine the susceptibility of porcine trophoblast cell line (PTr2) to PRRSV-2 infection to understand the potential role of the trophoblast in viral transmission to fetuses in vivo., Methods: PTr2 cells were exposed in vitro to PRRSV-2 and then subjected to immunofluorescence analysis (IF), flow cytometry (FCM), real-time quantitative PCR (RT-qPCR), transmission electron microscopy (TEM) and immunogold electron microscopy (IEM) to assess viral infection. The effects of PRRSV-2 on PTr2 cell cycle progression and apoptosis, as well as the ability of PTr2 cells to produce infectious viral particles were also examined., Results: PRRSV-2 was readily detected in PTr2 cells by IF, FCM, RT-qPCR, TEM and IEM techniques. RT-qPCR and FCM results of a time course of infection of PTr2 cells indicated PRRSV-2 load decreased over time after initial infection up to 72 h. PRRSV-2 infection altered PTr2 cell cycle with a selective increase of cells within the G2/M phase and also induced apoptosis. TEM and IEM demonstrated PRRSV-2 within and on the surface of PTr2 cells and PRRSV-2 virions released from PTr2 cells infected naïve MARC-145 cells inducing cytopathic effects., Discussion: Trophoblast cells are susceptible to PRRSV-2 infection and release live virions capable of inducing cytopathic effects in naïve cells. This suggests a possible mechanism by which PRRSV-2 can breach the MFI resulting in fetal infection and death., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mucohaemorrhagic diarrhea associated with Brachyspira hampsonii infection has emerged as a production-limiting disease in western Canada. This pathogen was first described in North America in 2010, and reports of its detection occurred concurrently in western Canada and the United States. Since that time, Brachyspira hampsonii has been detected in Europe, both in pigs and in waterfowl. The origin of B. hampsonii and the timing and reasons for its emergence are unknown. We conducted a retrospective study of historic, archived cases of porcine colitis to determine when B. hampsonii was first evident in western Canada. A total of 206 samples from 114 cases submitted from 57 different farms or productions systems in 1984 and 1999-2009 were screened using real-time PCR assays targeting B. hampsonii genomovars I and II, and Brachyspira hyodysenteriae (the traditional agent of swine dysentery). In most cases, positive real-time PCR results were confirmed by amplification and sequencing of additional gene targets. A total of 9, 7 and 5 samples tested positive for B. hampsonii (I), B. hampsonii (II) or B. hyodysenteriae respectively. The results of this study push the date of first appearance of B. hampsonii in pigs in western Canada back to 2002 (B. hampsonii (I)) and 2006 (B. hampsonii (II)), which is up to 7 years before the new species were first identified in fresh samples., (© 2018 Blackwell Verlag GmbH.)

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.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe reproductive failure characterized by high fetal morbidity and mortality leading to substantial economic losses to the swine industry. Evaluation of spatiotemporal transmission of PRRSV at the maternal-fetal interface (MFI) is critical for understanding fetal infection. Localization of PRRSV-2 strain NVSL 97-7895 at different regions of the MFI in 20 pregnant gilts at 2, 5, 8, 12 and 14 days post-inoculation (dpi) were analyzed by immunofluorescence (IF). Samples of MFI were collected from 15 inoculated and 5 control gilts and transplacental PRRSV transmission assessed in randomly selected fetuses from each litter. Localization of NVSL 97-7895 antigen immunoreactivity in the MFI was focused in three major areas: endometrial connective tissues (ENDO), the feto-maternal junction (FMJ) and fetal placenta (PLC). NVSL 97-7895 was detected at the FMJ by 2 dpi. At 2, 5 and 8 dpi, NVSL 97-7895 was localized within the ENDO and FMJ, whereas at 12 and 14 dpi, it was mainly localized in the PLC. Using a novel IF strategy for counting and size sorting NVSL 97-7895 viral antigen in situ, results of this study indicate that non-cell-associated mechanisms are involved in PRRSV transmission across the MFI., (© FEMS 2018.)

Swine are a key reservoir host for influenza A viruses (IAVs), with the potential to cause global pandemics in humans. Gaps in surveillance in many of the world's largest swine populations impede our understanding of how novel viruses emerge and expand their spatial range in pigs. Although US swine are intensively sampled, little is known about IAV diversity in Canada's population of ~12 million pigs. By sequencing 168 viruses from multiple regions of Canada, our study reveals that IAV diversity has been underestimated in Canadian pigs for many years. Critically, a new H1 clade has emerged in Canada (H1α-3), with a two-amino acid deletion at H1 positions 146-147, that experienced rapid growth in Manitoba's swine herds during 2014-2015. H1α-3 viruses also exhibit a higher capacity to invade US swine herds, resulting in multiple recent introductions of the virus into the US Heartland following large-scale movements of pigs in this direction. From the Heartland, H1α-3 viruses have disseminated onward to both the east and west coasts of the United States, and may become established in Appalachia. These findings demonstrate how long-distance trading of live pigs facilitates the spread of IAVs, increasing viral genetic diversity and complicating pathogen control. The proliferation of novel H1α-3 viruses also highlights the need for expanded surveillance in a Canadian swine population that has long been overlooked, and may have implications for vaccine design.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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.

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.

Over a period of 5 years, 10 pure-bred Boxer puppies, 9-16 weeks old, were presented with a history of sudden death and were diagnosed with pyogranulomatous myocarditis. The myocarditis was characterized by a mixed infiltrate composed predominantly of neutrophils and macrophages. In our retrospective study, original case records and archived materials were examined. All dogs were positive for Borrelia burgdorferi on immunohistochemistry (IHC). There was no evidence of infectious agents in formalin-fixed, paraffin-embedded (FFPE) heart tissue sections stained with hematoxylin and eosin, Ziehl-Neelsen, Gram, Grocott methenamine silver, Warthin-Starry, Von Kossa, and Steiner-Chapman stains. IHC for Chlamydia sp., Toxoplasma gondii, Neospora caninum, West Nile virus, and canine parvovirus also yielded a negative result in all dogs. Polymerase chain reaction testing for vector-borne pathogens on heart tissue from 9 of the dogs (1 frozen and 8 FFPE samples) yielded positive results for 1 dog with B. burgdorferi as well as Anaplasma phagocytophilum in another dog. Subsequently, 2 additional cases were found in a French Bulldog and a French Bulldog-Beagle mix that had identical morphology, test results, age, and seasonality to these 10 Boxer dogs. The similarities in the seasonality, signalment of the affected dogs, and the gross and microscopic lesions suggest a common etiology. Positive IHC and morphologic similarities to human Lyme carditis indicate that B. burgdorferi is likely the agent involved. An additional consideration for these cases is the possibility of a breed-specific autoimmune myocarditis or potential predisposition for cardiopathogenic agents in young Boxers., (© 2016 The Author(s).)

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.

Influenza viruses are a common cause of respiratory disease in swine. Infections range in severity from asymptomatic to causing significant morbidity. The main objective of this study was to compare lung transcriptomic and epigenetic responses to influenza infection in pigs from high or low birth weight litters. The latter is a potential indicator of intrauterine growth restriction, a significant risk factor for prenatal programming effects. Individual pigs from high (HBW) or low birth weight (LBW) litters (n = 17) were inoculated with influenza A virus and euthanized 48 hours later. Lesion severity and viral loads were assessed as previously described. The transcriptional response to infection in LBW and HBW groups (n = 16) was assessed by microarray. A separate analysis of pigs classified as 'Resilient' (RES) or 'Susceptible' (SUS) (n = 6) on the basis of severity of lung pathology was also conducted. Eight genes were confirmed as differentially expressed for the birth weight comparison, including three antiviral genes with lower expression in LBW: ISG15, OAS1, and OAS2 (P<0.05). The promoter region methylation status of these three genes was assessed for each birth weight group, and no differences were found. These expression data are consistent with our previous finding that LBW pigs had less severe lesion scores and a trend towards lower viral titres in lung than the HBW cohort. The SUS v RES comparison identified 91 differentially expressed genes (FDR<0.05) that were enriched with functional annotation terms and pathways associated with inflammation. The cytokine genes IL6, IL8, and CCL2 were all upregulated in SUS pigs, and may have driven disease severity in these animals. In conclusion, this study found no evidence that the transcriptional immune response to influenza was adversely affected by low litter birth weight, but did identify several candidate genes for driving disease pathology.

Mechanisms of reproductive failure resulting from infection with porcine reproductive and respiratory syndrome virus (PRRSV) are still poorly understood. Presented herein are the results of a side-by-side evaluation of the pathogenicity of three type 2 PRRSV strains in a reproductive model, from a pilot study used to develop experimental conditions and laboratory methods for a larger experiment. Pregnant gilts were experimentally infected with PRRSV at gestation day 85 or served as uninfected negative controls. After 21 days, all gilts and fetuses were necropsied. Clinical signs, litter outcome, viral load, cytokine levels, and pathology were compared from samples collected among pigs exposed to the three PRRSV strains. Based on differences in histologic lesions, and fetal weights, and numeric differences in gilt serum cytokine levels, litter outcome and virus replication in fetal tissues KS06-483 appeared less virulent than NVSL 97-7895 and KS06-72109 isolates. Levels of chemokine ligand 2 (CCL2), interferon alpha (IFNα), and interferon gamma (IFNγ) were increased in PPRRSV-infected compared to non-infected gilts (0.01 > P < 0.06). Inoculation with NVSL 97-7895 induced higher levels of all three cytokines. All three PRRSV isolates were able to induce high mean viral load in individual litters, which was closely related to the proportion of PRRSV positive fetuses in the litter. Viral load in fetal samples was also positively associated with viral load at the maternal-fetal interface. All but one dead fetus were positive for PRRSV RNA, and higher concentrations of PRRSV RNA in fetal thymus increased the odds of fetal death. Our results suggest that virus replication in fetal tissues and the maternal-fetal interface, but not in other gilt tissues, are important for the outcome of reproductive PRRS. Additionally, our data indicate that umbilical lesions decreased corresponding to the use of pentobarbital sedation prior to euthanasia of pregnant gilts by captive bolt., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The complex and unresolved evolutionary origins of the 2009 H1N1 influenza pandemic exposed major gaps in our knowledge of the global spatial ecology and evolution of influenza A viruses in swine (swIAVs). Here we undertake an expansive phylogenetic analysis of swIAV sequence data and demonstrate that the global live swine trade strongly predicts the spatial dissemination of swIAVs, with Europe and North America acting as sources of viruses in Asian countries. In contrast, China has the world's largest swine population but is not a major exporter of live swine, and is not an important source of swIAVs in neighbouring Asian countries or globally. A meta-population simulation model incorporating trade data predicts that the global ecology of swIAVs is more complex than previously thought, and the United States and China's large swine populations are unlikely to be representative of swIAV diversity in their respective geographic regions, requiring independent surveillance efforts throughout Latin America and Asia.

Unlabelled: The capacity of influenza A viruses to cross species barriers presents a continual threat to human and animal health. Knowledge of the human-swine interface is particularly important for understanding how viruses with pandemic potential evolve in swine hosts. We sequenced the genomes of 141 influenza viruses collected from North American swine during 2002 to 2011 and identified a swine virus that possessed all eight genome segments of human seasonal A/H3N2 virus origin. A molecular clock analysis indicates that this virus--A/sw/Saskatchewan/02903/2009(H3N2)--has likely circulated undetected in swine for at least 7 years. For historical context, we performed a comprehensive phylogenetic analysis of an additional 1,404 whole-genome sequences from swine influenza A viruses collected globally during 1931 to 2013. Human-to-swine transmission occurred frequently over this time period, with 20 discrete introductions of human seasonal influenza A viruses showing sustained onward transmission in swine for at least 1 year since 1965. Notably, human-origin hemagglutinin (H1 and H3) and neuraminidase (particularly N2) segments were detected in swine at a much higher rate than the six internal gene segments, suggesting an association between the acquisition of swine-origin internal genes via reassortment and the adaptation of human influenza viruses to new swine hosts. Further understanding of the fitness constraints on the adaptation of human viruses to swine, and vice versa, at a genomic level is central to understanding the complex multihost ecology of influenza and the disease threats that swine and humans pose to each other., Importance: The swine origin of the 2009 A/H1N1 pandemic virus underscored the importance of understanding how influenza A virus evolves in these animals hosts. While the importance of reassortment in generating genetically diverse influenza viruses in swine is well documented, the role of human-to-swine transmission has not been as intensively studied. Through a large-scale sequencing effort, we identified a novel influenza virus of wholly human origin that has been circulating undetected in swine for at least 7 years. In addition, we demonstrate that human-to-swine transmission has occurred frequently on a global scale over the past decades but that there is little persistence of human virus internal gene segments in swine., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: "Brachyspira hampsonii", discovered in North America in 2010 associated with dysentery-like illness, is an economically relevant swine pathogen resulting in decreased feed efficiency and increased morbidity, mortality and medication usage. "B. hampsonii" clade II strain 30446 has been shown to be causally associated with mucohemorrhagic diarrhea and colitis. Our objectives were to determine if "Brachyspira hampsonii" clade I strain 30599 is pathogenic to pigs, and to evaluate the relative diagnostic performance of three ante mortem sampling methodologies (direct PCR on feces, PCR on rectal GenoTube Livestock swabs, Brachyspira culture from rectal swabs). Five-week old pigs were intragastrically inoculated thrice with 108 genomic equivalents "B. hampsonii" (n = 12), or served as sham controls (n = 6). Feces were sampled and consistency assessed daily. Necropsies were performed 24 h after peak clinical signs., Results: One pig died due to unrelated illness. Nine of 11 inoculated pigs, but no controls, developed mucoid or mucohemorrhagic diarrhea (MHD). Characteristic lesions of swine dysentery were observed in large intestine. "B. hampsonii" strain 30599 DNA was detected by qPCR in feces of all inoculated pigs for up to 6 days prior to the onset of MHD. The organism was isolated from the feces and colons of pigs demonstrating MHD, but not from controls. B. intermedia was isolated from inoculated pigs without MHD, and from 5 of 6 controls., Conclusions: We conclude that "Brachyspira hampsonii" clade I strain 30599 is pathogenic and causes mucohemorrhagic diarrhea and colitis in susceptible pigs. Moreover, the three sampling methodologies performed similarly. GenoTube Livestock, a forensic swab designed to preserve DNA during shipping is a useful tool especially in settings where timely transport of diagnostic samples is challenging.

In spite of extensive research, the mechanisms of reproductive disease associated with Porcine Reproductive and Respiratory Syndrome virus (PRRSv) are still poorly understood. The objectives of this large scale study were to evaluate associations between viral load and fetal preservation, determine the impact of type 2 PRRSv on fetal weights, and investigate changes in ORF5 PRRSv genome in dams and fetuses during a 21-day period following challenge. At gestation day 85 (±1), 114 gilts were experimentally infected with type 2 PRRSv, while 19 gilts served as reference controls. At necropsy, fetuses were categorized according to their preservation status and tissue samples were collected. PRRSv RNA concentrations were measured in gilt serum collected on days 0, 2, 6, and 21 post-infection, as well as in gilt and fetal tissues collected at termination. Fetal mortality was 41±22.8% in PRRS infected litters. Dead fetuses appeared to cluster in some litters but appeared solitary or random in others. Nine percent of surviving piglets were meconium-stained. PRRSv RNA concentration in fetal thymus, fetal serum and endometrium differed significantly across preservation category and was greatest in tissues of meconium-stained fetuses. This, together with the virtual absence of meconium staining in non-infected litters indicates it is an early pathological condition of reproductive PRRS. Viral load in fetal thymus and in fetal serum was positively associated with viral load in endometrium, suggesting the virus exploits dynamic linkages between individual maternal-fetal compartments. Point mutations in ORF5 sequences from gilts and fetuses were randomly located in 20 positions in ORF5, but neither nucleotide nor amino acid substitutions were associated with fetal preservation. PRRSv infection decreased the weights of viable fetuses by approximately 17%. The considerable variation in gilt and fetal outcomes provides tremendous opportunity for more detailed investigations of potential mechanisms and single nucleotide polymorphisms associated with fetal death.

Detection of influenza A virus (IAV), viral antigen, nucleic acid, or antibodies in swine is dependent upon the collection of the appropriate sample type, the quality of the sample, and the proper storage and handling of the sample. The diagnostic tests to be performed should be considered prior to sample collection. Sera are acceptable samples for ELISA or hemagglutination inhibition tests, but not for real-time RT-PCR. Likewise, swabs and/or tissues are acceptable for real-time RT-PCR and virus isolation. The sample type will also depend on the age of swine that are being tested; oral fluids can be successfully collected from weaned pigs usually greater than 3 weeks of age, whereas nasal swabs should be collected from suckling pigs in the first weeks of life. This chapter outlines the collection of different specimen types and procedures for proper specimen handling.

Background: Epigenetic studies have shown that low-birth-weight (LBW) and growth restriction has been associated with reduced immune function in humans and reduced passive immunity in pigs. To examine the immune responses of high-birth-weight (HBW) and LBW groups of pigs, influenza A virus infection was used as an exemplifier of neonatal respiratory disease., Objectives: The objectives of this study were (i) to compare clinical, immunological, and pathological outcome of influenza infection in HBW to LBW pigs and (ii) to establish standardized sampling sites, score each site independently with set criteria, and compare scores between sites., Methods: Sixty-eight 4-week-old pigs originating from either HBW or LBW litters were intratracheally inoculated with 10(6.3) TCID50 /ml of A/swine/Texas/4199-2/1998 H3N2 and euthanized 48 hours later. Samples were collected 2.5 cm from the tip of both cranial and middle lung lobes. The formalin-fixed paraffin-embedded tissue sections were scored in a blinded manner by a single pathologist using established scoring criteria for routine and immunohistochemical stains. Clinical parameters, lung and nasal swab virus titers, and cytokine levels for interferon-alpha and interleukin-1-beta, IL-6, and IL-8 were measured., Results and Conclusions: Lung lesion severity and influenza staining intensity were significantly lower in LBW compared with HBW pigs (P < 0.05). Additionally, examining just the LBW group, the significant difference between lobes (P = 0.009) showed that the mean score for the right cranial lung lobe was higher compared with the other three lobes., (© 2013 Blackwell Publishing Ltd.)

Brachyspira is associated with diarrhea and colitis in pigs, and control of these pathogens is complicated by their complex ecology. Identification of wildlife reservoirs of Brachyspira requires the discrimination of colonized animals and those simply contaminated through environmental exposure. Lesser snow geese (Chen caerulescens caerulescens) were sampled in the Canadian arctic during the summer of 2011, and cloacal swabs were cultured on selective media. Brachyspira isolates were obtained from 15/170 (8.8 %) samples, and 12/15 isolates were similar to isolates previously recovered from pigs, including "Brachyspira hampsonii", a recently characterized species associated with dysentery-like disease in pigs in North America. A pilot inoculation study with one strongly β-hemolytic B. hampsonii isolate resulted in fecal shedding of the isolate by inoculated pigs for up to 14 days post-inoculation, but no severe clinical disease. Results of this study indicate that lesser snow geese can be colonized by Brachyspira strains that can also colonize pigs. Millions of lesser snow geese (C. caerulescens caerulescens) travel through the major pork-producing areas of Canada and the USA during their annual migration, making them a potential factor in the continental distribution of these bacteria.

The binding of influenza A viruses to epithelial cells in the respiratory tract of mammals is a key step in the infection process. Therefore, direct assessment of virus-host cell interaction using virus histochemistry (VH) will enhance our understanding of the pathogenesis of these new viruses. For this study, the authors selected viruses that represented the 4 main genetic clusters of North American swine H1 (SwH1) viruses, along with A/California/04/2009 H1N1 and a vaccine strain for the positive controls, and the virus label, fluorescein isothiocyanate (FITC), for the negative control. A group of 5 viruses containing a 2-amino acid insertion adjacent to the binding site of the hemagglutinin protein and their presumed ancestral viruses were also examined for changes in binding patterns. Viruses were bound to formalin-fixed paraffin-embedded, 6-week-old (6w) and adult pig tissues. Qualitative VH scores per respiratory zone ranged from + to +++, with bronchioles having the highest and most consistent scores, regardless of animal age. For the 6w bronchioles, a quantitative VH score was calculated using digital images of 5 bronchioles per tissue section using image analysis software. Significant differences in attachment were found among the SwH1 viruses (P < .0001) and among the ancestral and insertion viruses (P < .0001). These results provide new insights on virus binding to porcine respiratory epithelial cells and the usefulness of morphometric scores. The results also highlight limitations of in vitro techniques, including VH for predicting virulence and host range.

The commonality of influenza A virus (IAV) exposure and vaccination on swine farms in the United States ensures that the majority of neonatal pigs will have some degree of maternal immunity to IAV. The influence of maternal immunity on IAV transmission in neonatal pig populations will impact virus prevalence and infection dynamics across pig populations. The main objective of this study was to assess the impact of maternally derived immunity on IAV transmission in an experimental setting. Neonatal pigs suckled colostrum and derived maternal (passive) immunity from sows in one of three treatment groups: (a) non-vaccinated control (CTRL) or vaccinated with (b) homologous (PASSV-HOM) or (c) heterologous (PASSV-HET) inactivated experimental IAV vaccines. Sentinel neonatal pigs derived from the groups above were challenged with IAV via direct contact with an experimentally infected pig (seeder pig) and monitored for IAV infection daily via nasal swab sampling. A susceptible-infectious-recovered (SIR) experimental model was used to obtain and estimate transmission parameters in each treatment group via a generalized linear model. All sentinel pigs in the CTRL (30/30) and PASSV-HET (30/30) groups were infected with IAV following contact with the seeder pigs and the reproduction ratio estimates (95% confidence interval) were 10.4 (6.6-15.8) and 7.1 (4.2-11.3), respectively. In contrast, 1/20 sentinel pigs in the PASSV-HOM group was infected following contact with the seeder pigs and the reproduction ratio estimate was significantly lower compared to the CTRL and PASSV-HET groups at 0.8 (0.1-3.7). Under the conditions of this study, IAV transmission was reduced in neonatal pigs with homologous maternal immunity compared to seronegative neonatal pigs and pigs with heterologous maternal immunity as defined in this study. This study provides estimates for IAV transmission in pigs with differing types of maternal immunity which may describe the influence of maternal immunity on IAV prevalence and infection dynamics in pig populations., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Influenza A virus vaccines currently contain a mixture of isolates that reflect the genetic and antigenic characteristics of the currently circulating strains. This study was conducted to evaluate the efficacy of a trivalent inactivated swine influenza virus vaccine (Flusure XP) in pigs challenged with a contemporary α-cluster H1N1 field isolate of Canadian swine origin. Pigs were allocated to vaccinated, placebo, and negative-control groups and monitored for respiratory disease for 5 d after challenge. On the challenge day and 5 d after challenge the serum of the vaccinated pigs had reciprocal hemagglutination inhibition antibody titers 40 for all the vaccine viruses but ≤ 20 for the challenge virus. Gross lesions were present in the lungs of all pigs that had been inoculated with the challenge virus, but the proportion of lung tissue consolidated did not differ significantly between the placebo and vaccinated pigs. However, the amount of virus was significantly reduced in the nasal secretions, lungs, and bronchoalveolar lavage fluid in the vaccinated pigs compared with the placebo pigs. These results indicate that swine vaccinated with Flusure XP were partially protected against experimental challenge with a swine α-cluster H1N1 virus that is genetically similar to viruses currently circulating in Canadian swine.

Revealing the frequency and determinants of reassortment among RNA genome segments is fundamental to understanding basic aspects of the biology and evolution of the influenza virus. To estimate the extent of genomic reassortment in influenza viruses circulating in North American swine, we performed a phylogenetic analysis of 139 whole-genome viral sequences sampled during 1998-2011 and representing seven antigenically distinct viral lineages. The highest amounts of reassortment were detected between the H3 and the internal gene segments (PB2, PB1, PA, NP, M and NS), while the lowest reassortment frequencies were observed among the H1γ, H1pdm and neuraminidase segments, particularly N1. Less reassortment was observed among specific haemagglutinin-neuraminidase combinations that were more prevalent in swine, suggesting that some genome constellations may be evolutionarily more stable.

Porcine oral fluids have been used for the detection of Porcine reproductive and respiratory syndrome virus and Porcine circovirus-2. The objective of the present study was to determine whether Influenza A virus (FLUAV) is present in porcine oral fluids at detectable levels and to validate a standard FLUAV molecular diagnostic test for porcine oral fluids. Pen-based oral fluid samples were collected on 3, 4, 5, and 6 days postinfection (DPI) from 4 groups of 6 pigs each that were inoculated intratracheally with A/Swine/Iowa/00239/2004 H1N1 and from 2 untreated or mock-inoculated groups of 6 pigs each that served as negative controls. Individual nasal swabs were also collected from these 36 pigs on 3 and 7 DPI. All oral fluid samples were examined for the presence of FLUAV by matrix gene real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and virus isolation. Nasal swabs were tested initially by virus isolation followed by retest of negative samples with real-time RT-PCR. No oral fluid sample from virus-inoculated pigs was positive by virus isolation, but 15 of 16 positive (94%) oral fluids were positive by real-time RT-PCR. In contrast, virus was isolated from 32 of 48 (67%) nasal swabs collected from virus-inoculated pigs. In addition, 382 of 910 porcine oral fluids collected from pigs in the field between August 1, 2009, and January 31, 2010, were positive by real-time RT-PCR. The results of the present study indicate that pen-based oral fluids provide an easy, effective, and safe collection method for the detection of FLUAV with rapid testing methods such as real-time RT-PCR.