TLR9 mediates IgA production in the porcine small intestine during PEDV infection.

IgA plays a vital role in defending against the infectious pathogens. However, the specific regulatory pathways involved in IgA secretion in the context of PEDV infection have remained elusive. Therefore, in this study, we explore the molecular mechanisms underlying IgA secretion in response to infection, with a particular focus on PEDV, a devastating enteric virus affecting global swine production. Our investigation begins by examining changes in IgA concentrations in both serum and small intestinal contents following PEDV infection in 2- and 4-week-old pigs. Remarkably, a significant increase in IgA levels in these older pigs post-infection were observed. To delve deeper into the regulatory mechanisms governing IgA secretion in response to PEDV infection, isolated porcine intestinal B cells were co-cultured with monocytes derived DCs (Mo-DCs) in vitro. In the intestinal DC-B cell co-cultures, IgA secretion was found to increase significantly after PEDV infection, as well as upregulating the expression of AID, GLTα and PSTα reflecting isotype switching to IgA. In addition, the expression of TLR9 was upregulated in these cultures, as determined by RT-qPCR and western blotting. Moreover, our findings extend to in vivo observations, where we detected higher levels of TLR9 expression in the ileum of pig post PEDV infection. Collectively, our results highlight the ability of PEDV to stimulate the generation of IgA, particularly in elder pigs, and identify TLR9 as a critical mediator of IgA production within the porcine intestinal microenvironment during PEDV infection.

Identification of four genes responsible for antimicrobial resistance of MEL-B against S. aureus.

There is increasing interest in the antimicrobial activity of mannosylerythritol lipids-B (MEL-B) against Gram-positive bacteria such as Staphylococcus aureus (S. aureus). However, the specific molecules involved in MEL-B's antimicrobial action against S. aureus have not been identified. This study utilized the Nebraska transposon mutant library (NTML), which contains 1920 mutants, each lacking three-quarters of the genes found in S. aureus. The NTML was screened to identify mutants resistant to MEL-B. Four mutants (Accession Number: SAUSA300_0904, SAUSA300_0752, SAUSA300_0387, and SAUSA300_2311) largely unaffected by incubation with MEL-B, indicating MEL-B resistance. Despite the strong binding of MEL-B to these mutants, the four molecules encoded by the deleted genes (yjbI, clpP, pbuX, or brpS) in each mutant were not directly recognized by MEL-B. Given that these molecules are not localized on the outer surface of S. aureus and that the antibacterial activity of MEL-B against S. aureus is facilitated by the effective transfer of two antibacterial fatty acids (caprylic acid and myristoleic acid) to S. aureus via ME, the deletion of each of the four molecules may alter the peptidoglycan structure, potentially inhibiting the effective transfer of these antimicrobial fatty acids into S. aureus.

Linoleic acid: a natural feed compound against porcine epidemic diarrhea disease.

IMPORTANCE: Porcine epidemic diarrhea virus (PEDV) is a pig coronavirus that causes severe diarrhea and high mortality in piglets, but as no effective drugs are available, this virus threatens the pig industry. Here, we found that the intestinal contents of specific pathogen-free pigs effectively blocked PEDV invasion. Through proteomic and metabolic analyses of the intestinal contents, we screened 10 metabolites to investigate their function and found that linoleic acid (LA) significantly inhibited PEDV replication. Further investigations revealed that LA inhibited viral replication and release mainly by binding with PEDV NSP5 to regulate the PI3K pathway and, in particular, inhibiting AKT phosphorylation. In vivo experiments illustrated that orally administered LA protected pigs from PEDV challenge and severe diarrhea. These findings provide strong support for exploring antiviral drugs for coronavirus treatment.

TGF-ß1 reduces the differentiation of porcine IgA-producing plasma cells by inducing IgM+ B cells apoptosis via Bax/Bcl2-Caspase3 pathway.

Transforming growth factor ß1 (TGF-ß1) performs a critical role in maintaining homeostasis of intestinal mucosa regulation and controls the survival, proliferation, and differentiation of many immune cells. In this study, we discovered that the infection of porcine epidemic diarrhea virus (PEDV), a coronavirus, upregulated TGF-ß1 expression via activating Tregs. Besides, recombinant porcine TGF-ß1 decreased the percentage of CD21+ B cells within the lymphocyte population in vitro. We further found that TGF-ß1 reduced the IgA-secreting B cell numbers and also inhibited plasma cell differentiation. Additional investigations revealed that TGF-ß1 induced the apoptosis of IgM+ B cells in both peyer's patches (PPs) and peripheral blood (PB) through the activation of the Bax/Bcl2-Caspase3 pathway. Conversely, the application of the TGF-ß1 signaling inhibitor SB431542 significantly antagonized the TGF-ß1-induced reduction of IgA secretion and B cell apoptosis and restored plasma cell differentiation. Collectively, TGF-ß1 plays an important role in regulating the survival and differentiation of porcine IgA-secreting B cells through the classical mitochondrial apoptosis pathway. These findings will facilitate future mucosal vaccine designs that target the regulation of TGF-ß1 for the control of enteric pathogens in the pig industry.

Perforin and granzyme A release as novel tool to measure NK cell activation in chickens.

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs including spleen and gut, where they play an important role in the defense against infections. Interaction of NK cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin (PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an additional tool to study activity of cytotoxic lymphocytes in chickens.

Mini-review: microbiota have potential to prevent PEDV infection by improved intestinal barrier.

Porcine epidemic diarrhea virus (PEDV) infection poses a significant threat to the global pig industry. Current prevention and control strategies are inadequate in protecting pigs from new PEDV variants. This review aims to examine the relationship between PEDV and intestinal microbes, and investigate whether modulating intestinal microbes could affect PEDV infection. The mechanisms by which various intestinal microbes affect viral infection were initially introduced. Intestinal microbes can influence enteric viral infection through direct contact, such as binding, or by affecting interferons (IFNs) production and the intestinal barrier. Influencing the intestinal barrier by microbes can impact PEDV infection in young piglets. To narrow down the range of microbes that may influence PEDV infection, this review summarized microbes that change after infection. Short chain fatty acids (SCFAs), bacterial cell components, and toxins from microbes were identified as important mediators affecting PEDV infection. SCFAs primarily strengthen the intestinal barrier and inhibit intestinal inflammation, while bacterial cell components and toxins are more likely to damage the intestinal barrier. Therefore, this review hypothesizes that fecal transplantation, which allows the host to colonize more SCFAs-producing microbes, may prevent PEDV infection. However, these hypotheses require further proof, and the transplantation of intestinal microbes in pigs requires more exploration.

Linoleic acid stimulation results in TGF-ß1 production and inhibition of PEDV infection in vitro.

Linoleic acid (LA) is recommended to improve pork quality. However, whether it affects the intestinal immune response in pigs is still unclear. Our ex vivo experiments demonstrated that LA stimulation resulted in increased frequencies of Tregs in PBMCs but not in Peyer's Patches (PPs). The results of RT-qPCR, flow cytometry, and ELISA indicated that LA increased the TGF-ß1 expression level in DCs isolated from PEDV infected pigs. Furthermore, RT-qPCR and flow cytometry results demonstrated that TGF-ß1 was associated with higher frequencies of Tregs both in PBMCs and PPs. Additional investigations showed that TGF-ß1 inhibited PEDV infection in vitro. Besides, knocking-out TGF-ß1 in IPEC-J2 cells resulted in higher viral load. Taken together, our results demonstrated that LA stimulation resulted in enhanced production of TGF-ß1 by DC, which resulted in higher frequencies of Tregs production and inhibition of PEDV infection.

Oral administration of Lactic acid bacteria inhibits PEDV infection in young piglets.

Since the emergence of the highly pathogenic porcine epidemic diarrhea virus (PEDV) strain in 2010, the prevention of porcine epidemic diarrhea (PED) in pig farms remains problematic. To find the reasons behind the high mortality in young piglets, the relative mRNA expression of inflammation-related factors in infected pigs of different ages as well as uninfected pigs were detected by RT-qPCR. The results showed that the mRNA expression of these factors including IL-6 and TNF-α was more increased in infected younger piglets than infected older pigs. To clarify the relationship between these inflammation related factors, the pairwise linear correlation between the relative expression of these factors were analyzed and showed as network mapping with different correlation coefficients. A strong positive correlation was observed between the expression of various factors in 1-week-old piglets. Combined with the difference in mortality of PEDV infection in pigs of different ages, we hypothesized that lactic acid bacteria (LAB) could inhibit PEDV infection in newborn piglets, and an in vivo experiment was carried out. The results of survival rate and wet/dry ratio showed that LAB alleviated PEDV indued mortality and diarrhea. The detection of viral copies and tissue section staining showed less observed viruses in LAB treated pig. RT-qPCR results of gene expression in intestines showed that LAB modulated the gene expression of various host barrier genes, indicating that LAB is potential to inhibit PEDV infection by regulating the host intestinal barrier. However, to use LAB as therapy, how to improve the efficiency on inhibiting PEDV infection needs further studies.

Pigs' intestinal barrier function is more refined with aging.

The high mortality upon enteric virus infection in piglets causes huge economic losses. To control these infections, potential causes for this high susceptibility for enteric virus infections in younger piglets were analyzed by comparing the intestinal barrier between 1-week, 2-week and 4-week-old piglets. In this study, histological staining was used to analyze morphological differences in intestinal villi, real-time qPCR was performed to assess mRNA expression levels of genes that were related to viral infection and differentiation of immune cells, and flow cytometry was utilized to measure the frequencies of T cells. According to the results obtained, 1-week-old piglets have intestinal villi with shallower crypts, less well developed epithelial cells and a more immature immune system compared to older pigs. Moreover, high amounts of enteric virus invasion-assisting proteins but low amounts of resistant proteins in 1-week piglets could also be a reason for the high susceptibility of 1-week-old piglets.

Chicken-derived RSPO1 and WNT3 contribute to maintaining longevity of chicken intestinal organoid cultures.

Intestinal organoids are advanced cellular models, which are widely used in mammalian studies to mimic and study in vivo intestinal function and host-pathogen interactions. Growth factors WNT3 and RSPO1 are crucial for the growth of intestinal organoids. Chicken intestinal organoids are currently cultured with mammalian Wnt3a and Rspo1, however, maintaining their longevity has shown to be challenging. Based on the limited homology between mammalian and avian RSPO1, we expect that chicken-derived factors are required for the organoid cultures. Isolated crypts from embryonic tissue of laying hens were growing in the presence of chicken WNT3 and RSPO1, whereas growth in the presence of mammalian Wnt3a and Rspo1 was limited. Moreover, the growth was increased by using Prostaglandin E2 (PGE2) and a Forkhead box O1-inhibitor (FOXO1-inhibitor), allowing to culture these organoids for 15 passages. Furthermore, stem cells maintained their ability to differentiate into goblets, enterocytes and enteroendocrine cells in 2D structures. Overall, we show that chicken intestinal organoids can be cultured for multiple passages using chicken-derived WNT3 and RSPO1, PGE2, and FOXO1-inhibitor.

Transgenerational Effects of Maternal Immune Activation on Specific Antibody Responses in Layer Chickens.

Activation of the maternal immune system may affect innate and adaptive immune responses in the next generation and may therefore have implications for vaccine efficacy and dietary immune modulation by feed additives. However, transgenerational effects on immune responses in chickens have been investigated to a limited extend. The present study investigated effects of intratracheal (i.t) specific and aspecific immune activation of laying hens on specific antibody production in the next generation. In two experiments laying hens received intratracheally an immune stimulus with human serum albumin (HuSA) or lipopolysaccharide (LPS). In experiment 1, hatchlings of the immune activated hens were at 4 weeks i.t. immunized with HuSA or HuSA+LPS. Maternal immune activation with LPS increased HuSA specific IgY and IgM responses in offspring. These results suggest a transgenerational effect of the maternal immune system on the specific antibody response in the next generation. In experiment 2 hatchlings received either ß-glucan-enriched feed or control feed and were i.t. immunized with HuSA. Maternal immune activation with LPS decreased IgY anti-HuSA responses after HuSA immunization within hatchlings that received ß-glucan enriched feed. The results of Experiment 2 suggest a transgenerational link between the innate immune system of mother and specific antibody responses in offspring. Despite variabilities in the outcomes of the two experiments, the observations of both suggest a link between the maternal innate immune system and the immune system of the offspring. Furthermore, our results may imply that maternal activation of the innate immune system can influence immune modulating dietary interventions and vaccine strategies in the next generation.

Effects of E scherichia coli Nissle 1917 on the Porcine Gut Microbiota, Intestinal Epithelium and Immune System in Early Life.

Early in life and particularly around weaning, piglets are susceptible to infections because of abrupt social, environmental, and dietary changes. Dietary interventions with probiotic bacteria have gained popularity because of the increased awareness of the direct link between diet and health. In this study, piglets received the probiotic strain Escherichia coli Nissle 1917 (EcN) or a control treatment perorally from day 2 after birth until 2 weeks post-weaning. To investigate spatio-temporal effects of EcN on the gut microbiota composition, intestinal epithelial gene expression and immune system, feces, digesta, blood, scraping material and mesenteric lymph node tissue were collected at different time points. In addition, oral vaccinations against Salmonella enterica serovar Typhimurium were administered on days 21 and 45 of the study to assess the immunocompetence. EcN-treated pigs showed a reduced diversity of taxa within the phylum Proteobacteria and a lower relative abundance of taxa within the genus Treponema during the pre-weaning period. Moreover, EcN induced T cell proliferation and Natural Killer cell activation in blood and enhanced IL-10 production in ex vivo stimulated mesenteric lymph node cells, the latter pointing toward a more regulatory or anti-inflammatory state of the local gut-associated immune system. These outcomes were primarily observed pre-weaning. No significant differences were observed between the treatment groups with regards to body weight, epithelial gene expression, and immune response upon vaccination. Differences observed during the post-weaning period between the treatment groups were modest. Overall, this study demonstrates that the pre-weaning period offers a 'window of opportunity' to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation.

Long-chain glucomannan supplementation modulates immune responsiveness, as well as intestinal microbiota, and impacts infection of broiler chickens with Salmonella enterica serotype Enteritidis.

The zoonotic pathogen Salmonella enterica serotype Enteritidis (SE) causes severe disease in young chickens. Restriction on antibiotic use requires alternative SE control strategies such as nutritional solutions to improve the resistance of chickens. In this study, chickens were fed long-chain glucomannan (GM) or standard diet and challenged with SE at seven days of age. During 21 days post-infection (dpi), we determined numbers and responsiveness of natural killer (NK) and T cells in ileum and spleen, and SE-specific antibody titers in serum. Microbiota compositions in ileum and caeca were determined, as well as correlations of these with numbers and function of immune cells. Some of the samples in the control group had numerically higher CFUs than the GM-treated group. In addition, the relative abundance of SE based on DNA assessment was significantly lower at 21 dpi upon GM supplementation. At 3 dpi, numbers of intraepithelial NK cells were significantly higher, while activation of intraepithelial NK cells (7 dpi), numbers of intraepithelial cytotoxic CD8+ T cells (14 dpi) and SE-specific antibodies (14 dpi) were numerically higher. Furthermore, relative abundance of the commensal lactic acid bacteria (LAB) significantly increased with GM supplementation post-infection. Higher relative abundance of streptococci was associated with reduced SE in ileal and caecal contents at 21 dpi. Relative abundance of streptococci negatively correlated with SE counts and positively correlated with NK cell activation and SE-specific antibodies, which suggests involvement of the commensal LAB in NK cell responsiveness. These results indicate that GM supplementation modulates the immune system, intestinal microbiota and impacts SE infection of young chickens.

The Interplay between Salmonella and Intestinal Innate Immune Cells in Chickens.

Salmonellosis is a common infection in poultry, which results in huge economic losses in the poultry industry. At the same time, Salmonella infections are a threat to public health, since contaminated poultry products can lead to zoonotic infections. Antibiotics as feed additives have proven to be an effective prophylactic option to control Salmonella infections, but due to resistance issues in humans and animals, the use of antimicrobials in food animals has been banned in Europe. Hence, there is an urgent need to look for alternative strategies that can protect poultry against Salmonella infections. One such alternative could be to strengthen the innate immune system in young chickens in order to prevent early life infections. This can be achieved by administration of immune modulating molecules that target innate immune cells, for example via feed, or by in-ovo applications. We aimed to review the innate immune system in the chicken intestine; the main site of Salmonella entrance, and its responsiveness to Salmonella infection. Identifying the most important players in the innate immune response in the intestine is a first step in designing targeted approaches for immune modulation.

Transgenerational effects of innate immune activation in broiler breeders on growth performance and immune responsiveness.

The impact of transgenerational effects on growth performance and immunity has not yet been studied extensively within the poultry husbandry sector. An important factor is the impact of the hens on the physical well-being and fitness to the environment of the offspring. This study is the first to investigate the effect of stimulating the maternal innate immune system with lipopolysaccharides (LPS) or ß-glucan on growth performance and immune responses in the next generation. Transgenerational effects and consequences of these maternal treatments were further examined using a necrotic enteritis (NE) challenge model in the offspring. We show that offspring of LPS-treated broiler breeders have a higher feed efficiency from 14 to 21 days of age, that is, the period just after the NE challenge. Moreover, more broiler chickens with intestinal lesions after the NE challenge were found in the offspring of the LPS-treated broiler breeders. Both the LPS and ß-glucan maternal treatments resulted in transgenerational effects on blood-derived monocytes by showing a tendency of decreased IL1ß mRNA levels after ex vivo LPS stimulation. These data are a first indication that broiler breeder hens can affect immune responsiveness and feeding efficiency of their offspring in a transgenerational manner.

Overcoming scientific barriers in the transition from in vivo to non-animal batch testing of human and veterinary vaccines.

INTRODUCTION: Before release, vaccine batches are assessed for quality to evaluate whether they meet the product specifications. Vaccine batch tests, in particular of inactivated and toxoid vaccines, still largely rely on in vivo methods. Improved vaccine production processes, ethical concerns, and suboptimal performance of some in vivo tests have led to the development of in vitro alternatives. AREAS COVERED: This review describes the scientific constraints that need to be overcome for replacement of in vivo batch tests, as well as potential solutions. Topics include the critical quality attributes of vaccines that require testing, the use of cell-based assays to mimic aspects of in vivo vaccine-induced immune responses, how difficulties with testing adjuvanted vaccines in vitro can be overcome, the use of altered batches to validate new in vitro test methods, and how cooperation between different stakeholders is key to moving the transition forward. EXPERT OPINION: For safety testing, many in vitro alternatives are already available or at an advanced level of development. For potency testing, in vitro alternatives largely comprise immunochemical methods that assess several, but not all critical vaccine properties. One-to-one replacement by in vitro alternatives is not always possible and a combination of methods may be required.

A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens.

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens.

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

The contribution of the immune response to enhanced colibacillosis upon preceding viral respiratory infection in broiler chicken in a dual infection model.

Colibacillosis in chickens caused by avian pathogenic Escherichia coli (APEC) is known to be aggravated by preceding infections with infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV). The mechanism behind these virus-induced predispositions for secondary bacterial infections is poorly understood. Here we set out to investigate the immunopathogenesis of enhanced respiratory colibacillosis after preceding infections with these three viruses. Broilers were inoculated intratracheally with APEC six days after oculonasal and intratracheal inoculation with IBV, NDV, aMPV or buffered saline. After euthanasia at 1 and 8 days post infection (dpi) with APEC, birds were macroscopically examined and tissue samples were taken from the trachea, lungs and air sacs. In none of the groups differences in body weight were observed during the course of infection. Macroscopic lesion scoring revealed most severe tissue changes after NDV-APEC and IBV-APEC infection. Histologically, persistent tracheitis was detected in all virus-APEC groups, but not after APEC-only infection. In the lungs, mostly APEC-associated transient pneumonia was observed. Severe and persistent airsacculitis was present after NDV-APEC and IBV-APEC infection. Bacterial antigen was detected by immunohistochemistry only at 1 dpi APEC, predominantly in NDV-APEC- and IBV-APEC-infected lungs. Higher numbers of CD4+ and CD8+ lymphocytes persisted over time in NDV-APEC- and IBV-APEC-infected tracheas, as did CD4+ lymphocytes in NBV-APEC- and IBV-APEC-infected air sacs. KUL01+ cells, which include monocytes and macrophages, and TCRγδ+ lymphocytes were observed mostly in lung tissue in all infected groups with transient higher numbers of KUL01+ cells over time and higher numbers of TCRγδ+ lymphocytes mainly at 8 dpi. qPCR analysis revealed mostly trends of transient higher levels of IL-6 and IFNγ mRNA in lung tissue after IBV-APEC and also NDV-APEC infection and persistent higher levels of IL-6 mRNA after aMPV-APEC infection. In spleens, transient higher levels of IL-17 mRNA and more persistent higher levels of IL-6 mRNA were observed after all co-infections. No changes in IL-10 mRNA expression were seen. These results demonstrate a major impact of dual infections with respiratory viruses and APEC, compared to a single infection with APEC, on the chicken respiratory tract and suggest that immunopathogenesis contributes to lesion persistence.