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.
The re-emergence of Brachyspira-associated disease in pigs since the late 2000s has illuminated some of the diagnostic challenges associated with this genus; notably, the lack of standardized ...antimicrobial susceptibility testing (AST) methods and interpretive criteria. Consequently, laboratories have relied heavily on highly variable in-house developed methods. There are currently no published investigations describing the antimicrobial susceptibility of Brachyspira isolates collected from pigs in Canada. The first objective of this study was therefore to develop a standardized protocol for conducting agar dilution susceptibility testing of Brachyspira spp., including determining the optimal standardized inoculum density, a key test variable that impacts test performance. The second objective was to determine the susceptibility of a collection of western Canadian Brachyspira isolates using the standardized methodology. After assessing multiple media, an agar dilution test was standardized in terms of starting inoculum (1-2 × 108 CFU/ml), incubation temperature and time, and assessed for repeatability. The antimicrobial susceptibility of a collection of clinical porcine Brachyspira isolates (n = 87) collected between 2009-2016 was then determined. This method was highly reproducible; repeat susceptibility testing yielded identical results 92% of the time. Although most of the isolates had very low MICs to the commonly used antimicrobials to treat Brachyspira-associated infections, several isolates with elevated MICs (>32 μg/ml) for tiamulin, valnemulin, tylosin, tylvalosin, and lincomycin were identified. Overall, this study underscores the importance of establishing CLSI approved clinical breakpoints for Brachyspira to facilitate the interpretation of test results and support the evidence-based selection of antimicrobials in swine industry.
Swine dysentery is causally associated with
Brachyspira hampsonii
and
B
.
hyodysenteriae
infection. Given the importance of transmission models in understanding re-emergent diseases and developing ...control strategies such as vaccines, the objective of this experiment was to evaluate two experimental natural transmission (seeder pig) models in grower pigs, each with 24 animals. Seeder pigs were intragastrically inoculated using broth cultures of either
B
.
hampsonii
strain 30446 (genomovar II) or
B
.
hyodysenteriae
strain G44. In trial 1, three seeder pigs were placed into two pens containing nine susceptible contact pigs creating a 1:3 seeder:contact ratio. This was sufficient to achieve natural
B
.
hampsonii
infection of 13/18 (72%) contact pigs, however, the incidence of mucoid or mucohemorrhagic diarrhea (MMHD) in contact pigs differed significantly between pens (4/9 versus 9/9;
P
= 0.03). In trial 2, eight seeder pigs inoculated intragastrically with
B
.
hampsonii
did not develop MMHD but when re-inoculated with
B
.
hyodysenteriae
14 days later, all developed mucohemorrhagic diarrhea within 13 days of re-inoculation. Two seeder pigs were placed into each of 4 contact pens each containing 4 pigs. This 1:2 seeder:contact ratio resulted in natural infection of 14/16 (87%) contact pigs with incubation period ranging from 9–15 days. There were no significant differences among pens in incubation period, duration, clinical period or severity of diarrhea. These trials demonstrated that a 1:2 seeder:contact ratio with groups of six grower pigs per pen sustained natural transmission of
B
.
hyodysenteriae
G44 with greater consistency in the incidence of MMHD among pens compared to a
B
.
hampsonii
30446 transmission model using 1:3 seeder:contact ratio in pens of 12. Understanding why
B
.
hampsonii
intragastric inoculation failed in one experiment warrants additional research.
Swine dysentery (SD) is a worldwide production-limiting disease of growing-finishing pigs in commercial farms. The importance of the large intestinal microbiota in the swine dysentery pathogenesis ...has been established, but not well characterized. The objective of this study was to characterize the fecal bacterial microbiota of pigs immediately prior to developing clinical signs of swine dysentery. A total of 60 fecal samples were collected from 15 pigs with SD. Sampling times included a time point prior to SD (d0, n=15), 2 days before mucohaemorrhagic diarrhea was observed (d-2SD, n=15), 1 day before mucohaemorrhagic diarrhea was observed (d-1SD, n=15), and the day when pigs developed mucohemorragic diarrhea (MHD, n=15). Sequencing of cpn60 amplicons was used to profile the microbiome, and analyses were performed on QIIME2. Increased Chao1 index in d-1SD and MHD samples when compared to the d0 was the only change observed in alpha diversity. No differences between sampling times on beta diversity (Bray-Curtis dissimilarity) were found. Although a small sample size was investigated, differential abundance analysis revealed that Alistipes dispar and Parabacteroides gordonii were increased in MHD fecal samples when compared to d-2SD and d-1SD. It is suggested that these taxa may play a role in the pathogenesis of SD, which is known to require the presence of Brachyspira spp. and an anaerobe for severe disease development.
Swine dysentery (SD) caused by pathogenic Brachyspira spp. is an economic challenge for the swine industry. In research settings, experimental reproduction of swine dysentery typically relies on ...intragastric inoculation which has shown variable success. This project aimed to improve the consistency of the experimental inoculation protocol used for swine dysentery in our laboratory. Over six experiments, we evaluated the influence of group housing in inoculated pigs using a frozen-thawed broth culture of strongly hemolytic B. hyodysenteriae strain D19 (Trial A), compared the relative virulence of B. hyodysenteriae strains D19 and G44 (Trial B), compared inoculum volumes (50 mL vs 100 mL) for G44 and B. hampsonii 30446 (Trial C), and performed three independent trials evaluating intragastric inoculation using different oral inoculation methods: oral feed balls (Trial D), and oral syringe bolus of 100 mL (Trial E) or 300 mL (Trial F). Intragastric inoculation with a fresh broth culture of B. hyodysenteriae strain G44 resulted in a shorter incubation period and a higher proportionate duration of mucohemorrhagic diarrhea (MMHD) compared to D19. Intragastric inoculation with either 50 or 100 mL of B. hampsonii 30446 or B. hyodysenteriae (G44) were statistically equivalent. Oral inoculation with 100 mL or 300 mL also yielded similar results to intragastric inoculation but was more expensive due to the additional work and supplies associated with syringe training. Our future research will use intragastric inoculation with 100 mL of a fresh broth culture containing B. hyodysenteriae strain G44 as it yields a high incidence of mucohaemorrhagic diarrhea with a reasonable cost.
Salmonella enterica serovar Typhimurium is an animal welfare and public health concern due to its ability to parasite livestock and potentially contaminate pork products. To reduce Salmonella ...shedding and the risk of pork contamination, antibiotic therapy is used and can contribute to antimicrobial resistance. Here we hypothesized that immune system education by the microbiota can play a role in intestinal resilience to infection. We used amoxicillin (15mg/Kg) to modulate the intestinal microbiome of 10 piglets, paired with same age pigs that received a placebo (n = 10) from 0 to 14 days of age. Animals were euthanized at 4-weeks old. Each pig donated colon sections for ex vivo culture (n = 20 explants/pig). Explants were inoculated with S. Typhimurium, PBS or LPS (n = 6 explants/pig/group, plus technical controls). The gut bacteriome was characterized by sequencing of the 16S rRNA at 7, 21 days of age, and upon in vitro culture. Explants response to infection was profiled through high-throughput mRNA sequencing. In vivo antibiotic treatment led to β-diversity differences between groups at all times (P<0.05), while α-diversity did not differ between amoxicillin and placebo groups on day 21 and at euthanasia (P<0.03 on day 7). Explant microbiomes were not different from in vivo. In vitro challenge with S. Typhimurium led to lower necrosis scores in explants from amoxicillin-treated pigs, when compared to explants placebo-treated pigs (P<0.05). This was coupled with the activation of immune-related pathways in explants from amoxicillin-treated pigs (IL-2 production, NO production, BCR activation), when compared to placebo-treated pigs. In addition, several DNA repair and intestinal wound healing pathways were also only activated in explants from amoxicillin-treated pigs. Taken together, these findings suggest that immune education by the amoxicillin-disturbed microbiota may have contributed to mitigate intestinal lesions following pathogen exposure.
Porcine reproductive and respiratory syndrome virus (PRRSV) infection during late gestation negatively affects fetal development. The objective of this study was to identify the fetal organs most ...severely impacted following infection, and evaluate the relationship between this response and fetal phenotypes. RNA was extracted from fetal heart, liver, lung, thymus, kidney, spleen, and loin muscle, collected following late gestation viral challenge of pregnant gilts. Initially, gene expression for three cell cycle promoters (CDK1, CDK2, CDK4) and one inhibitor (CDKN1A) were evaluated in biologically extreme phenotypic subsets including gestational age-matched controls (CON), uninfected (UNIF), high-viral load viable (HV-VIA), and high-viral load meconium-stained (HV-MEC) fetuses. There were no differences between CON and UNIF groups for any gene, indicating no impact of maternal infection alone. Relative to CON, high-viral load (HV-VIA, HV-MEC) fetuses showed significant downregulation of at least one CDK gene in all tissues except liver, while CDKN1A was upregulated in all tissues except muscle, with the heart and kidney most severely impacted. Subsequent evaluation of additional genes known to be upregulated following activation of P53 or TGFb/SMAD signaling cascades indicated neither pathway was responsible for the observed increase in CDKN1A. Finally, analysis of heart and kidney from a larger unselected population of infected fetuses from the same animal study showed that serum thyroxin and viral load were highly correlated with the expression of CDKN1A in both tissues. Collectively these results demonstrate the widespread suppression in cell division across all tissues in PRRSV infected fetuses and indicate a non-canonical regulatory mechanism.
A pregnant gilt infected with porcine reproductive and respiratory syndrome virus (PRRSV) can transmit the virus to her fetuses across the maternal-fetal-interface resulting in varying disease ...outcomes. However, the mechanisms leading to variation in fetal outcome in response to PRRSV infection are not fully understood. Our objective was to assess targeted immune-related gene expression patterns and pathways in the placenta and fetal thymus to elucidate the molecular mechanisms involved in the resistance/tolerance and susceptibility of fetuses to PRRSV2 infection. Fetuses were grouped by preservation status and PRRS viral load (VL): mock infected control (CTRL), no virus detected (UNINF), virus detected in the placenta only with viable (PLCO-VIA) or meconium-stained fetus (PLCO-MEC), low VL with viable (LVL-VIA) or meconium-stained fetus (LVL-MEC), and high VL with viable (HVL-VIA) or meconium-stained fetus (HVL-MEC).
The host immune response was initiated only in fetuses with detectable levels of PRRSV. No differentially expressed genes (DEG) in either the placenta or thymus were identified in UNINF, PLCO-VIA, and PLCO-MEC when compared to CTRL fetuses. Upon fetal infection, a set of core responsive IFN-inducible genes (CXCL10, IFIH1, IFIT1, IFIT3, ISG15, and MX1) were strongly upregulated in both tissues. Gene expression in the thymus is a better differentiator of fetal VL; the strong downregulation of several innate and adaptive immune pathways (e.g., B Cell Development) are indicative of HVL. Gene expression in the placenta may be a better differentiator of fetal demise than the thymus, based-on principle component analysis clustering, gene expression patterns, and dysregulation of the Apoptosis and Ubiquitination pathways.
Our data supports the concept that fetal outcome in response to PRRSV2 infection is determined by fetal, and more significantly placental response, which is initiated only after fetal infection. This conceptual model represents a significant step forward in understanding the mechanisms underpinning fetal susceptibility to the virus.
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) in late gestation causes a profound suppression of circulating maternal and fetal thyroid hormone during a critical window of ...development. To understand this relationship, we evaluated thyroid hormone metabolism at the maternal–fetal interface and within fetal tissues, along with hormone metabolite levels in serum. Fetuses were classified using an established model based on viral load in serum and thymus, and preservation status, including uninfected (UNIF), high-viral load viable (HV-VIA), and high-viral load meconium-stained (HV-MEC), with additional controls from sham-inoculated gilts (CON). Expression of three iodothyronine deiodinases, five sulfotransferases, sulfatase, and two solute carriers known to transport thyroid hormone were evaluated in maternal endometrium and fetal placenta, liver, and kidney. Serum thyroxin (T4), reverse triiodothyronine (rT3), and diiodothyronine (T2) were evaluated via liquid chromatography tandem mass spectrometry. Significant changes in gene expression were observed in all four tissues, with the liver being the most severely impacted. We observed local and fetal specific regulation of maternal tissues through significant upregulation of DIO2 and DIO3 expression in the endometrium corresponding to infected but viable fetuses relative to uninfected and control fetuses. Expression levels of DIO2 and DIO3 were significantly higher in the resilient (HV-VIA) fetuses relative to the susceptible (HV-MEC) fetuses. A substantial decrease in serum T4 was confirmed, with no corresponding increase in rT3 or T2. Collectively, these results show that thyroid hormone metabolism is altered at the maternal–fetal interface and within the PRRSV infected fetus and is associated with fetal viability.
To better understand the host response to porcine reproductive and respiratory virus-2 (PRRSV2) we evaluated circulating thyroid hormone and associated gene expression in a late gestation challenge ...model. Pregnant gilts were inoculated at gestation day 85 and fetal samples collected at either 12 or 21 days post-infection (dpi). A subset of fetuses was selected for analysis based on viability and viral load categorized as either uninfected-viable (UNIF), high viral load viable (HV-VIA) or high viral load meconium stained (HV-MEC) and were compared with gestational age matched controls (CON). In dams, circulating levels of total T3 and T4 decreased in the acute period following infection and rebounded by 21 dpi. A similar effect was observed in fetuses, but was largely restricted to HV-VIA and HV-MEC, with minimal decrease noted in UNIF relative to CON at 21 dpi. Gene expression in fetal heart at 12 dpi showed significant decompensatory transcription of thyroid hormone transporters (SLC16A2) and deiodinases (DIO2, DIO3), which was not observed in brain. Correspondingly, genes associated with cell cycle progression (CDK1,2,4) were downregulated in only the heart of highly infected fetuses, while expression of their inhibitor (CDKN1A) was upregulated in both tissues. Finally, expression of genes associated with cardiac stress including CAMKD and AGT were upregulated in the hearts of highly infected fetuses, and a shift in expression of MYH6 to MYH7 was observed in HV-MEC fetuses specifically. Collectively, the results suggest PRRSV2 infection causes a hypothyroid state that disproportionally impacts the fetal heart over the brain.
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