Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry.

Technical note: Validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli.

Mammary epithelial cells (MEC) participate in the first line of defense of the mammary gland to invading pathogens. In vitro culture of MEC is widely used as a model to study the capacity of these cells to sense and respond to mastitis-causing bacteria. Analysis of gene expression by quantitative PCR (qPCR) following exposure to bacteria or bacterial constituents is a powerful tool to assess responses of MEC to pathogens. Although internal standards such as reference genes are required for qPCR to yield valid data, the validation of proper genes to quantify mRNA transcripts in MEC exposed to pro-inflammatory stimuli has never been reported. In this study, 10 commonly used reference genes belonging to different functional classes (ACTB, ATP5B, EIF2B2, GAPDH, PPIA, SDHA, SUZ12, UXT, YWHAZ, and 18s rRNA) were analyzed by qPCR to determine the most stable in bovine MEC unstimulated and stimulated with mastitis pathogens (Staphylococcus aureus or Escherichia coli), microbial agonists of the innate immune system (lipoteichoic acid and muramyl dipeptide, or lipopolysaccharide), or proinflammatory cytokines (IL-17A and tumor necrosis factor-α). An M value was used as a measure of gene stability as determined using the geNorm application. This study demonstrated that the expression of the 10 reference genes was stable under the different experimental conditions. These data will be useful for bovine mastitis research in selecting reference genes and validating reverse transcription-qPCR data.

Effectiveness of two intranasal vaccines for the control of bovine respiratory disease in newborn beef calves: A randomized non-inferiority multicentre field trial.

Bovine respiratory syncytial virus (BRSV) and bovine parainfluenza-3 virus (bPI3V) are major causes of bovine respiratory disease (BRD) in newborn calves worldwide. Vaccination is widely used to prevent BRD, and intranasal vaccines for BRSV and bPI3V were developed to overcome interference from BRSV and bPI3V-specific maternally derived antibodies. Many experimental challenge trials have demonstrated that intranasal vaccines for BRSV and bPI3V are efficacious, but effectiveness under field conditions has been demonstrated less often, especially for newborn beef calves. The objective of this field trial was to compare the effectiveness of a newly available commercial BRSV-bPI3V intranasal vaccine with that of a benchmarked one in newborn beef calves reared in a cow-calf system. A total of 935 calves from 39 farms were randomized into two vaccine groups (Bovalto Respi Intranasal [Vaccine A], n=468; Rispoval RS+PI3 Intranasal [Vaccine B], n=467), and monitored during the in-house risk period up to three months after vaccination. Non-inferiority analysis was performed by calculating the difference in BRD prevalence between the two vaccine groups. No significant differences were observed between vaccines regarding clinical outcomes of morbidity, mortality, duration between vaccination and BRD occurrence, or treatments required. Because the upper limit of the 2-sided 95% confidence interval of the difference in BRD prevalence between the two treatment groups (0.8%) was less than the margin of non-inferiority (δ=5%), a non-inferiority of Vaccine A was concluded. In conclusion, Vaccine A is at least as effective as Vaccine B for the prevention of BRD in newborn beef cattle in a cow-calf system under field conditions.

Prospecting potential links between PRRSV infection susceptibility of alveolar macrophages and other respiratory infectious agents present in conventionally reared pigs.

Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is one of the main component of the porcine respiratory disease complex (PRDC), which strongly impact the pig production. Although PRRSV is often considered as a primary infection that eases subsequent respiratory coinfections, the possibility that other PRDC components may facilitate PRRSV infection has been largely overlooked. The main cellular targets of PRRSV are respiratory macrophages among them alveolar macrophages (AM) and pulmonary intravascular macrophages (PIM). AM, contrarily to PIM, are directly exposed to the external respiratory environment, among them co-infectious agents. In order to explore the possibility of a co-infections impact on the capacity of respiratory macrophages to replicate PRRSV, we proceed to in vitro infection of AM and PIM sampled from animals presenting different sanitary status, and tested the presence in the respiratory tract of these animals of the most common porcine respiratory pathogens (PCV2, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma floculare, Pasteurella multocida, Bordetella bronchiseptica, Streptoccocus suis). In this exploratory study with a limited number of animals, no statistic differences were observed between AM and PIM susceptibility to in vitro PRRSV infection, nor between AM coming from animals presenting very contrasting respiratory coinfection loads.

Induction of porcine regulatory cells by mycophenolic Acid-treated dendritic cells.

Tolerance induction in murine allogeneic transplantation is relatively easy, often by induction of regulatory T cells (Treg). Unfortunately, the implementation of these models in clinical situations has not yielded reliable protocols of tolerance induction in humans. Our project sought to create a preclinical model of tolerance induction in large animals. Our current efforts seek to induce and characterize porcine Treg, obtaining dendritic cells (DC) able to preferentially stimulate them. DCs were differentiated from blood monocytes with porcine recombinant interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 6 days. These DCs were then stimulated by human CD40 ligand-transfected L cells with or without mycophenolic acid (MPA) for 48 hours. We analyzed surface marker expression, cytokine synthesis, and ability to stimulate allogeneic peripheral blood mononuclear cells (PBMC). The porcine lymphocytes underwent 4 rounds of 1-week stimulation with allogeneic DC treated or not with MPA. At the end of this coculture we analyzed their capacity to suppress allogeneic PBMC proliferation induced by mature DC. Our results showed that porcine DCs pretreated with MPA display a low expression of B7 costimulatory molecules, produce low levels of IL-12, and induce weak proliferation of allogeneic lymphocytes. Moreover, after 4 rounds of stimulation with MPA-treated DCs, PBMCs were able to inhibit an alloreactive response. These preliminary results suggested induction of a regulatory T-cell population that we are currently seeking to characterize.

Immunome differences between porcine ileal and jejunal Peyer's patches revealed by global transcriptome sequencing of gut-associated lymphoid tissues.

The epithelium of the intestinal mucosa and the gut-associated lymphoid tissues (GALT) constitute an essential physical and immunological barrier against pathogens. In order to study the specificities of the GALT transcriptome in pigs, we compared the transcriptome profiles of jejunal and ileal Peyer's patches (PPs), mesenteric lymph nodes (MLNs) and peripheral blood (PB) of four male piglets by RNA-Seq. We identified 1,103 differentially expressed (DE) genes between ileal PPs (IPPs) and jejunal PPs (JPPs), and six times more DE genes between PPs and MLNs. The master regulator genes FOXP3, GATA3, STAT4, TBX21 and RORC were less expressed in IPPs compared to JPPs, whereas the transcription factor BCL6 was found more expressed in IPPs. In comparison between IPPs and JPPs, our analyses revealed predominant differential expression related to the differentiation of T cells into Th1, Th2, Th17 and iTreg in JPPs. Our results were consistent with previous reports regarding a higher T/B cells ratio in JPPs compared to IPPs. We found antisense transcription for respectively 24%, 22% and 14% of the transcripts detected in MLNs, PPs and PB, and significant positive correlations between PB and GALT transcriptomes. Allele-specific expression analyses revealed both shared and tissue-specific cis-genetic control of gene expression.

Chlamydia suis and Chlamydia trachomatis induce multifunctional CD4 T cells in pigs.

Chlamydia trachomatis infections are the most prominent bacterial sexually-transmitted disease world-wide and a lot of effort is put into the development of an effective vaccine. Pigs have been shown to be a valuable animal model for C. trachomatis vaccine development. The aim of this study was to decipher the T-cell-mediated immune response to chlamydial infections including C. trachomatis and C. suis, the chlamydia species naturally infecting pigs with a demonstrated zoonotic potential. Vaginal infection of pigs with C. suis and C. trachomatis lasted from 3 to 21days and intra-uterine infection was still present after 21days in 3 out of 5 C. suis- and 4 out of 5 C. trachomatis-inoculated animals and caused severe pathological changes. Humoral immune responses including neutralizing antibodies were found predominantly in response to C. suis starting at 14days post inoculation. The T-cell-mediated immune responses to C. trachomatis and C. suis-infections started at 7days post inoculation and consisted mainly of CD4+ T cells which were either IFN-γ single cytokine-producing or IFN-γ/TNF-α double cytokine-producing T-helper 1 cells. IL-17-producing CD4+ T cells were rare or completely absent. The T-cell-mediated immune responses were triggered by both homologous or heterologous re-stimulation indicating that cross-protection between the two chlamydia species is possible. Thus, having access to a working genital C. suis and C. trachomatis infection model, efficient monitoring of the host-pathogen interactions, and being able to accurately assess the responses to infection makes the pig an excellent animal model for vaccine development which also could bridge the gap to the clinical phase for C. trachomatis vaccine research.

Recombination in the alphaherpesvirus bovine herpesvirus 1.

Herpesviruses are DNA viruses characterized by a low rate of nucleotide substitution. Therefore, other mechanisms must be involved to their evolution, like recombination that can be seen as an essential evolutionary driving force of these viruses. Recombination contributes to the long-term evolution of alphaherpesviruses. It acts also to continuously create new alphaherpesvirus strains. We have used bovine herpesvirus 1 to investigate recombination both within DNA concatemers in infected cells and in vitro and in vivo at the end of the lytic cycle. The following results have been obtained: (i) intramolecular recombination occurs at the level of concatemers and gives rise to genomic segment inversions; (ii) intraspecific recombination occurs frequently both in vitro and in vivo; (iii) interspecific recombination is possible and requires two highly genetically related viruses; (iv) only simultaneous or closely separated infections lead to the production of recombinant viruses; (v) recombination between wild-type and glycoprotein defective vaccine virus can produce a glycoprotein defective virus keeping part of the virulence of parental wild-type virus. Recombination, by exchanging genomic segments, may modify the virulence of alphaherpesviruses. It must be carefully assessed for the biosafety of antiviral therapy, alphaherpesvirus-based vectors and live attenuated vaccines.

Extended semen for artificial insemination in swine as a potential transmission mechanism for infectious Chlamydia suis.

Although typically unnoticed, Chlamydia infections in swine have been shown to be both widespread and may impact production characteristics and reproductive performance in swine. Serum titers suggest Chlamydia infection within boar studs is common, and infected boars are known to shed chlamydia in their ejaculates. Although the transmission of viruses in chilled extended semen (ES) is well established, the inclusion of antibiotics in commercially available extender is generally believed to limit or preclude the transmission of infectious bacteria. The objective of this study was to evaluate the potential of ES used in artificial insemination to support transmission of the obligate intracellular bacteria Chlamydia suis (C suis) under standard industry conditions. First, the effect of C suis on sperm quality during storage was assessed by flow cytometry. Only concentrations above 5 × 10(5) viable C suis/mL caused significant spermicidal effects which only became evident after 7 days of storage at 17 °C. No significant effect on acrosome reaction was observed using any chlamydial concentration. Next, an in vitro infection model of swine testicular fibroblast cells was established and used to evaluate the effect of chilled storage on C suis viability under variable conditions. Storage in Androhep ES reduced viability by 34.4% at a multiplicity of infection of 1.25, an effect which increased to 53.3% when the multiplicity of infection decreased to 0.1. Interestingly, storage in semen extender alone (SE) or ES with additional antibiotics had no effect on bacterial viability. To rule out a secondary effect on extender resulting from metabolically active sperm, C suis was stored in fresh and expended SE and again no significant effect on bacterial viability was observed. Fluorescent microscopy of C suis in ES shows an association between bacteria and the remaining gel fraction after storage suggesting that the apparent reduction of bacterial viability in the presence of semen is due to adherence to gel fraction. Taken together, the results of this study suggest that C suis remains viable and infectious during chilled storage and is globally unaffected by antibiotics in extender. Thus, ES used in artificial insemination may act as a viable transmission mechanism for C suis in swine.

Biosafety of herpesvirus vectors.

Herpesviruses are large DNA viruses, which possess a number of advantages as gene delivery vectors. These relate to an ability to package large DNA insertions and establish lifelong latent infections in which the viral genome exists as a stable episome in the nucleus. For gene therapy to become a potential future treatment option, biosafe therapeutically efficient gene transfer is a central, but more and more stringent requirement. This review highlights the progress in development of herpesvirus based vectors, describes their properties as wall as discusses the biosafety concerns that are associated with their use in gene therapy. Thought was also given to biosafety issues pertaining to design and production of herpesvirus vector systems in therapeutic gene delivery.

The porcine innate immune system: an update.

Over the last few years, we have seen an increasing interest and demand for pigs in biomedical research. Domestic pigs (Sus scrofa domesticus) are closely related to humans in terms of their anatomy, genetics, and physiology, and often are the model of choice for the assessment of novel vaccines and therapeutics in a preclinical stage. However, the pig as a model has much more to offer, and can serve as a model for many biomedical applications including aging research, medical imaging, and pharmaceutical studies to name a few. In this review, we will provide an overview of the innate immune system in pigs, describe its anatomical and physiological key features, and discuss the key players involved. In particular, we compare the porcine innate immune system to that of humans, and emphasize on the importance of the pig as model for human disease.

In vitro and ex vivo analyses of co-infections with swine influenza and porcine reproductive and respiratory syndrome viruses.

Viral respiratory diseases remain problematic in swine. Among viruses, porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV), alone or in combination, are the two main known contributors to lung infectious diseases. Previous studies demonstrated that experimental dual infections of pigs with PRRSV followed by SIV can cause more severe disease than the single viral infections. However, our understanding of the impact of one virus on the other at the molecular level is still extremely limited. Thus, the aim of the current study was to determine the influence of dual infections, compared to single infections, in porcine alveolar macrophages (PAMs) and precision cut lung slices (PCLS). PAMs were isolated and PCLS were acquired from the lungs of healthy 8-week-old pigs. Then, PRRSV (ATCC VR-2385) and a local SIV strain of H1N1 subtype (A/Sw/Saskatchewan/18789/02) were applied simultaneously or with 3h apart on PAMs and PCLS for a total of 18 h. Immuno-staining for both viruses and beta-tubulin, real-time quantitative PCR and ELISA assays targeting various genes (pathogen recognition receptors, interferons (IFN) type I, cytokines, and IFN-inducible genes) and proteins were performed to analyze the cell and the tissue responses. Interference caused by the first virus on replication of the second virus was observed, though limited. On the host side, a synergistic effect between PRRSV and SIV co-infections was observed for some transcripts such as TLR3, RIG-I, and IFNß in PCLS. The PRRSV infection 3h prior to SIV infection reduced the response to SIV while the SIV infection prior to PRRSV infection had limited impact on the second infection. This study is the first to show an impact of PRRSV/SIV co-infection and superinfections in the cellular and tissue immune response at the molecular level. It opens the door to further research in this exciting and intriguing field.