C.A. Gagnon's research while affiliated with Université de Montréal and other places

This study evaluated the impact of different dietary vitamin D sources combined or not with vitamin C on serum 25(OH)D3 concentrations and the expression of genes related to antimicrobial peptides and to the metabolism of vitamin D, vitamin C, calcium (Ca), and phosphorus (P). From weaning (d 21) until d 68, 27 piglets were fed a post-weaning diet supplemented with: 1,500 IU of vitamin D3 (CTR; n = 9); 200 IU of vitamin D3 + 2,000 IU of 25(OH)D3 (HD; n = 8); or HD + 500 mg/kg of vitamin C (HD+C; n = 10). From d 40 to 68 piglets were exposed to porcine reproductive and respiratory syndrome (PRRS) using a natural disease challenge model. At d 68, after blood collection, all piglets were euthanized for the collection of lung and kidney samples. The concentrations of serum 25(OH)D3 were determined by HPLC and the mRNA expression of genes of interest was assessed by qPCR. Data were analyzed in a one-way analysis of variance with three treatments. At d 40 (after 19 d of supplementation), serum 25(OH)D3 concentrations were greatest for HD+C (22.48 ± 1.25 ng/mL), intermediate for HD (17.43 ± 0.84 ng/mL), and least for CTR (8.54 ± 0.65 ng/mL) treatments (P < 0.01). At d 68 (after 28 d of PRRS challenge) these values were greatest for HD (14.64 ± 2.77 ng/mL), intermediate for CTR (6.22 ± 0.59 ng/mL), and least for HD+C (2.18 ± 0.13 ng/mL; P < 0.01). No treatments effect was detected on pulmonary CYP27B1, RXRα, SLC23A1, RIGI, PR-39, and pBD1. In lungs, VDR expression was least for HD+C (P ≤ 0.06) whereas MDA5 expression was greatest for HD+C (P ≤ 0.07), and SLC23A2 expression was greatest for HD (P ≤ 0.09; Table 1). No treatments effect was detected on expression of renal FGFR1, KL, RXRα, SLC23A1, SLC23A2, and TPRV5. In kidney, CYP27B1 expression was greatest for HD+C groups (P = 0.03) and CYP24A expression was greatest for HD (P = 0.01). Renal EGR1 and NPT2c expressions were greatest for CTR groups and least for HD+C (P = 0.09) whereas WNK4 expression was greatest for HD+C and least for CTR (P = 0.10). Renal VDR expression was greatest for HD groups and least for HD+C (P ≤ 0.05). In conclusion, vitamin C supplementation increased serum 25(OH)D3 concentrations in pre-challenged piglets. These concentrations were reduced by vitamin C supplementation after PRRS challenge, which may be related to a greater synthesis of the active form of vitamin D in the kidney. Little effects were detected on pulmonary antimicrobial peptides, but the gene expression related to renal P reabsorption was influenced by HD+C whereas Ca reabsorption was apparently not impacted by treatments.

Infectious bronchitis virus (IBV) is a highly contagious Gammacoronavirus causing moderate to severe respiratory infection in chickens. Understanding the initial antiviral response in the respiratory mucosa is crucial for controlling viral spread. We aimed to characterize the impact of IBV Delmarva (DMV)/1639 and IBV Massachusetts (Mass) 41 at the primary site of infection, namely, in chicken tracheal epithelial cells (cTECs) in vitro and the trachea in vivo. We hypothesized that some elements of the induced antiviral responses are distinct in both infection models. We inoculated cTECs and infected young specific pathogen-free (SPF) chickens with IBV DMV/1639 or IBV Mass41, along with mock-inoculated controls, and studied the transcriptome using RNA-sequencing (RNA-seq) at 3 and 18 h post-infection (hpi) for cTECs and at 4 and 11 days post-infection (dpi) in the trachea. We showed that IBV DMV/1639 and IBV Mass41 replicate in cTECs in vitro and the trachea in vivo, inducing host mRNA expression profiles that are strain- and time-dependent. We demonstrated the different gene expression patterns between in vitro and in vivo tracheal IBV infection. Ultimately, characterizing host–pathogen interactions with various IBV strains reveals potential mechanisms for inducing and modulating the immune response during IBV infection in the chicken trachea.

Porcine circovirus 3 (PCV3) has been detected in pigs worldwide and associated with several clinical signs. To investigate the genetic diversity of PCV3 strains circulating in Canada, 44 PCV3 positive samples from Saskatchewan (2/44), Manitoba (2/44), Quebec (4/44), Alberta (11/44) and Ontario (25/44) submitted to diagnostic laboratories in Canada between 2019 and 2021 were sequenced and analyzed. Phylogenetic analysis of capsid genes showed that all of the 44 Canadian strains classified into PCV3a and segregated into seven lineages with common amino acid changes observed at A24V, R27K, N56D, T77S, Q98R, L150I (F) and R168K positions. Future studies are required to determine whether the polymorphisms in capsid proteins, as revealed in this study, could be associated with differences in the pathogenicity or antigenicity of PCV3 strains. This is the first phylogenetic analysis of PCV3 strains among different provinces in Canada.

The effects of porcine circovirus type 2b (PCV2b) and porcine reproductive and respiratory syndrome virus (PRRSV) co-infection in epithelial cells of the swine respiratory tract is unknown. In the present study, the newborn pig trachea cell line NPTr-CD163, which is permissive to both viruses, was persistently infected with PCV2b and then with PRRSV. Viral replication, cell viability, cytokines’ mRNA expression, and modulation of cellular genes expression were evaluated in infected cells. In NPTr-CD163 co-infection model, PCV2b replication was enhanced while PRRSV replication was suppressed. Cell viability was significantly decreased during PCV2b single infection and co-infection compared to mock-infected and PRRSV single infected cells. However, no difference was observed in cell viability between PCV2b and PCV2b/PRRSV infected cells. The IL6, IL8 and IL10 mRNA expression was significantly higher in co-infected cells compared to PCV2b and PRRSV single infected cells. Moreover, the IFN-α/β expression was significantly reduced in co-infected cells compared to PCV2b infected cells whereas it remained higher compared to PRRSV infected cells. The differential gene expression analysis revealed that the mRNA expression level of the cellular gene DUSP1 was significantly higher in all PRRSV infection models compared to PCV2b single infected cells. Knockdown of DUSP1 expression in co-infected cells significantly reduced PCV2b replication, suggesting a role for DUSP1 in PCV2b/PRRSV pathogenesis.

The pathogenesis of porcine circovirus type 2b (PCV2b) and swine influenza A virus (SwIV) during co-infection in swine respiratory cells is poorly understood. To elucidate the impact of PCV2b/SwIV co-infection, newborn porcine tracheal epithelial cells (NPTr) and immortalized porcine alveolar macrophages (iPAM 3D4/21) were co-infected with PCV2b and SwIV (H1N1 or H3N2 genotype). Viral replication, cell viability and cytokine mRNA expression were determined and compared between single-infected and co-infected cells. Finally, 3'mRNA sequencing was performed to identify the modulation of gene expression and cellular pathways in co-infected cells. It was found that PCV2b significantly decreased or improved SwIV replication in co-infected NPTr and iPAM 3D4/21 cells, respectively, compared to single-infected cells. Interestingly, PCV2b/SwIV co-infection synergistically up-regulated IFN expression in NPTr cells, whereas in iPAM 3D4/21 cells, PCV2b impaired the SwIV IFN induced response, both correlating with SwIV replication modulation. RNA-sequencing analyses revealed that the modulation of gene expression and enriched cellular pathways during PCV2b/SwIV H1N1 co-infection is regulated in a cell-type-dependent manner. This study revealed different outcomes of PCV2b/SwIV co-infection in porcine epithelial cells and macrophages and provides new insights on porcine viral co-infections pathogenesis.

The pathogenesis of porcine circovirus type 2b (PCV2b) and swine influenza A virus (SwIV) during co-infection in swine respiratory cells is poorly understood. To elucidate the impact of PCV2b/SwIV co-infection, newborn porcine tracheal epithelial cells (NPTr) and immortalized porcine alveolar macrophages (iPAM 3D4/21) were co-infected with PCV2b and SwIV (H1N1 or H3N2 genotype). Viral replication, cell viability and cytokine mRNA expression were determined and compared between single-infected and co-infected cells. Finally, 3’mRNA sequencing was performed to identify the modulation of gene expression and cellular pathways in co-infected cells. It was found that PCV2b significantly decreased and improved SwIV replication, in co-infected NPTr and iPAM 3D4/21 cells respectively, compared to single infected cells. Interestingly, PCV2b/SwIV co-infection synergistically up-regulated IFN expression in NPTr cells whereas in iPAM 3D4/21 cells, PCV2b impaired the SwIV IFN induced response, both correlating with SwIV replication modulation. RNA-sequencing analyses revealed that the modulation of gene expression and enriched cellular pathways during PCV2b/SwIV H1N1 co-infection is regulated in a cell type-dependent-manner. This study revealed different outcomes of PCV2b/SwIV co-infection in porcine epithelial cells and macrophages and provides new insights on porcine viral co-infections pathogenesis.

Platelet concentrate (PC) is an alternative therapy to treat mastitis in dairy cattle and is an alternative treatment for reproduction problems such as endometritis. Unfortunately, double-centrifugation processing methods described are time-consuming, require specialized laboratory equipment, and are usually done in a heterologous way, which risks herd health. To overcome this limitation, we evaluated single-step bovine PC processing methods readily applicable to a farm setting using an autologous conditioned plasma (ACP) production system. We investigated the hematologic findings, cytokines, and growth factors of the obtained PC samples. Autologous conditioned plasma was prepared using whole blood (WB) from 4 cows (group 1) using single-step centrifugation and 16 different processing methods. The 2 protocols that yielded the highest ratio of platelet to white blood cell (WBC) concentration were ACP-1 [720 × g (2,200 rpm), 5 min] and ACP-2 [929 × g (2,500 rpm), 3 min]. They were subsequently reproduced and compared using WB from 8 cows (group 2). Hematologic findings were quantified, IL-1β (cytokine) and growth factors [platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, bovine fibroblast growth factor (b-FGF)] were measured, and enrichment factors were compared between samples and processing methods. Hematological characteristics and platelet enrichment varied markedly among tested protocols and all were statistically different from WB. Protocol ACP-2 resulted in significantly greater platelet enrichment (mean 169% of WB) than ACP-1 (125% of WB). We found no significant difference between the 2 ACP preparation protocols with regard to leukocyte reduction (7.53–9.75% WBC compared with WB) or growth factor enrichment (124–125% PDGF, 95–100% TGF-β, 102–104% b-FGF, and 56–74% IL-1β compared with WB). In conclusion, both ACP protocols yielded a platelet concentration shown to promote healing for clinical applications in cattle, and the ACP-2 protocol resulted in a greater degree of platelet enrichment. Therefore, this protocol could be used for ACP production for clinical applications in cattle.

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), occurs sporadically in poultry flocks in Canada. Live attenuated chicken embryo origin (CEO) vaccines are being used routinely to prevent and control ILTV infections. However, ILT outbreaks still occur since vaccine strains could revert to virulence in the field. In this study, 7 Canadian ILTV isolates linked to ILT outbreaks across different time in Eastern Canada (Ontario; ON and Quebec; QC) were whole genome sequenced. Phylogenetic analysis confirmed the close relationship between the ON isolates and the CEO vaccines, whereas the QC isolates clustered with strains previously known as CEO revertant and wild-type ILTVs. Recombination network analysis of ILTV sequences revealed clear evidence of historical recombination between ILTV strains circulating in Canada and other geographical regions. The comparison of ON CEO clustered and QC CEO revertant clustered isolates with the LT Blen® CEO vaccine reference sequence showed amino acid differences in 5 and 12 open reading frames (ORFs), respectively. Similar analysis revealed amino acid differences in 32 ORFs in QC wild-type isolates. Compared to all CEO vaccine strains in the public domain, the QC wild-type isolates showed 15 unique mutational sites leading to amino acid changes in 13 ORFs. Our outcomes add to the knowledge of the molecular mechanisms behind ILTV genetic variance and provide genetic markers between wild-type and vaccine strains.

Introduction Porcine circovirus 3 (PCV3) has been detected in pigs worldwide and associated with several clinical signs. Methods To investigate the genetic diversity of PCV3 strains circulating in Canada, 44 PCV3 positive samples from Saskatchewan (2/44), Manitoba (2/44), Quebec (4/44), Alberta (11/44) and Ontario (25/44) submitted to diagnostic laboratories in Canada between 2019 and 2021 were sequenced and analyzed. Results Phylogenetic analysis of capsid genes showed that all of the 44 Canadian strains classified into PCV3a and segregated into seven lineages with common amino acid changes observed at A24V, R27K, N56D, T77S, Q98R, L150I (F) and R168K positions. Conclusion Future studies are required to determine whether the polymorphisms in capsid proteins, as revealed in this study, could be associated with differences in the pathogenicity or antigenicity of PCV3 strains. This is the first phylogenetic analysis of PCV3 strains among different provinces in Canada.