Influence of Immune Status on the Airborne Colonization of Piglets with Methicillin-Resistant Staphylococcus aureus (MRSA) Clonal Complex (CC) 398

Colonized vertebrates including humans and pigs are to date the main reservoirs of livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA). Currently, the mechanisms underlying colonization of pigs are not fully understood. We investigated the influence of piglet pre-immune status on airborne MRSA colonization. Three groups of MRSA-negative piglets were primed and exposed to airborne LA-MRSA (104 colony forming units (cfu)/m3) in an aerosol chamber for 24 h. One group was treated intramuscularly with dexamethasone (1 mg/kg body weight) to imitate weaning stress. The second group was exposed to bacterial endotoxin containing MRSA aerosol. Both conditions play a role in the development of multifactorial diseases and may promote MRSA colonization success. The third group served as control. The piglets' MRSA status was monitored for 21 days via swab samples. At necropsy, specific tissues and organs were analyzed. Blood was collected to examine specific immunological parameters. The duration of MRSA colonization was not extended in both treated groups compared to the control group, indicating the two immune-status influencing factors do not promote MRSA colonization. Blood sample analysis confirmed a mild dexamethasone-induced immune suppression and typical endotoxin-related changes in peripheral blood. Of note, the low-dose dexamethasone treatment showed a trend of increased MRSA clearance

CD4+ Th immunogenicity of the Ascaris spp. secreted products

Ascaris spp. is a major health problem of humans and animals alike, and understanding the immunogenicity of its antigens is required for developing urgently needed vaccines. The parasite-secreted products represent the most relevant, yet complex (>250 proteins) antigens of Ascaris spp. as defining the pathogen-host interplay. We applied an in vitro antigen processing system coupled to quantitative proteomics to identify potential CD4+ Th cell epitopes in Ascaris-secreted products. This approach considerably restricts the theoretical list of epitopes using conventional CD4+ Th cell epitope prediction tools. We demonstrate the specificity and utility of our approach on two sets of candidate lists, allowing us identifying hits excluded by either one or both computational methods. More importantly, one of the candidates identified experimentally, clearly demonstrates the presence of pathogen-reactive T cells in healthy human individuals against these antigens. Thus, our work pipeline identifies the first human T cell epitope against Ascaris spp. and represents an easily adaptable platform for characterization of complex antigens, in particular for those pathogens that are not easily amenable for in vivo experimental validation

The Inflammatory Response to Enterotoxigenic E. coli and Probiotic E. faecium in a Coculture Model of Porcine Intestinal Epithelial and Dendritic Cells

The gut epithelium constitutes an interface between the intestinal contents and the underlying gut-associated lymphoid tissue (GALT) including dendritic cells (DC). Interactions of intestinal epithelial cells (IEC) and resident DC are characterized by bidirectional crosstalk mediated by various factors, such as transforming growth factor-β (TGF-β) and thymic stromal lymphopoietin (TSLP). In the present study, we aimed (1) to model the interplay of both cell types in a porcine in vitro coculture consisting of IEC (cell line IPEC-J2) and monocyte-derived DC (MoDC) and (2) to assess whether immune responses to bacteria are altered because of the interplay between IPEC-J2 cells and MoDC. With regard to the latter, we focused on the inflammasome pathway. Here, we propose caspase-13 as a promising candidate for the noncanonical inflammasome activation in pigs. We conducted challenge experiments with enterotoxigenic Escherichia coli (ETEC) and probiotic Enterococcus faecium (E. faecium) NCIMB 10415. As potential mediators of IEC/DC interactions, TGF-β and TSLP were selected for analyses. Cocultured MoDC showed attenuated ETEC-induced inflammasome-related and proinflammatory interleukin (IL)-8 reactions compared with MoDC monocultures. Caspase-13 was more strongly expressed in IPEC-J2 cells cocultured with MoDC and upon ETEC incubation. We found that IPEC-J2 cells and MoDC were capable of releasing TSLP. The latter cells secreted greater amounts of TSLP when cocultured with IPEC-J2 cells. TGF-β was not modulated under the present experimental conditions in either cell types. We conclude that, in the presence of IPEC-J2 cells, porcine MoDC exhibited a more tolerogenic phenotype, which might be partially regulated by autocrine TSLP production. Noncanonical inflammasome signaling appeared to be modulated in IPEC-J2 cells. Our results indicate that the reciprocal interplay of the intestinal epithelium and GALT is essential for promoting balanced immune responses

Trilateral Relationship: Ascaris, Microbiota, and Host Cells

Ascariasis is a globally spread intestinal nematode infection of humans and a considerable concern in pig husbandry. Ascaris accomplishes a complex body migration from the intestine via the liver and lung before returning to the intestine. Tissue migration and the habitat shared with a complex microbial community pose the question of how the nematode interacts with microbes and host cells from various tissues. This review addresses the current knowledge of the trilateral relationship between Ascaris, its microbial environment, and host cells, and discusses novel approaches targeting these interactions to combat this widespread infection of livestock and man

Ascaris suum excretory/secretory products differentially modulate porcine dendritic cell subsets

Helminths produce excretory/secretory products (E/S) which can modulate the immune responses of their hosts. Dendritic cells (DC) are essential for initiating the host T cell response and are thus potential targets for modulation by helminth E/S. Here we study immunomodulation of porcine peripheral blood DC subsets following ex vivo stimulation with E/S from Ascaris suum, a common helminth of pigs with considerable public health and economic importance. Our data showed that the relative frequencies of DC subsets in porcine blood differ, with plasmacytoid DC (pDC) being the most prominent in healthy 6-month-old pigs. pDC are an important cytokine source, and we found that A. suum E/S suppressed production of the type 1 cytokines IL-12p40 and TNF-α by this subset following toll-like receptor (TLR) ligation. In contrast, conventional DC (cDC) are more efficient antigen presenters, and the expression of CD80/86, costimulatory molecules essential for efficient antigen presentation, were modulated differentially by A. suum E/S between cDC subsets. CD80/86 expression by type 1 cDC (cDC1) following TLR ligation was greatly suppressed by the addition of A. suum E/S, while CD80/86 expression by type 2 cDC (cDC2) was upregulated by A. suum E/S. Further, we found that IFN-γ production by natural killer (NK) cells following IL-12 and IL-18 stimulation was suppressed by A. suum E/S. Finally, in the presence of E/S, IFN-γ production by CD4+ T cells co-cultured with autologous blood-derived DC was significantly impaired. Together, these data provide a coherent picture regarding the regulation of type 1 responses by A. suum E/S. Responsiveness of pDC and cDC1 to microbial ligands is reduced in the presence of E/S, effector functions of Th1 cells are impaired, and cytokine-driven IFN-γ release by NK cells is limited

Age-dependent rise in IFN-γ competence undermines effective type 2 responses to nematode infection

The efficient induction of type 2 immune responses is central to the control of helminth infections. Previous studies demonstrated that strong Th1 responses driven by intracellular pathogens as well as a bias for type 1 activity in senescent mice impedes the generation of Th2 responses and the control of intestinal nematode infections. Here, we show that the spontaneous differentiation of Th1 cells and their expansion with age restrains type 2 immunity to infection with the small intestinal nematode H. polygyrus much earlier in life than previously anticipated. This includes the more extensive induction of IFN-γ competent, nematode-specific Th2/1 hybrid cells in BALB/c mice older than three months compared to younger animals. In C57BL/6 mice, Th1 cells accumulate more rapidly at steady state, translating to elevated Th2/1 differentiation and poor control of parasite fitness in primary infections experienced at a young age. Blocking of early IFN-γ and IL-12 signals during the first week of nematode infection leads to sharply decreased Th2/1 differentiation and promotes resistance in both mouse lines. Together, these data suggest that IFN-γ competent, type 1 like effector cells spontaneously accumulating in the vertebrate host progressively curtail the effectiveness of anti-nematode type 2 responses with rising host age

How, where and when to screen for porcine cytomegalovirus (PCMV) in donor pigs for xenotransplantation

Porcine cytomegalovirus (PCMV), that is actually a porcine roseolovirus (PRV), is a common herpesvirus in domestic pigs and wild boars. In xenotransplantation, PCMV/PRV has been shown to significantly reduce the survival time of pig kidneys and hearts in preclinical trials with different non-human primates. Furthermore, PCMV/PRV has been transmitted in the first pig to human heart xenotransplantation and contributed to the death of the patient. Although transmitted to the recipient, there is no evidence that PCMV/PRV can infect primate cells including human cells. PCMV/PRV is closely related to the human herpesviruses 6 and 7, and only distantly related to the human CMV (HCMV). Antiviral drugs used for the treatment of HCMV are less effective against PCMV/PRV. However, there are well described strategies to eliminate the virus from pig facilities. In order to detect the virus and to eliminate it, highly sensitive detection methods and the knowledge of how, where and when to screen the donor pigs is required. Here, a comparative testing of organs from pigs of different ages using polymerase chain reaction (PCR)-based and immunological methods was performed. Testing young piglets, PCMV/PRV was detected effectively by PCR in blood, bronchoalveolar lavage fluid, tonsils and heart. In adult animals, detection by PCR was not successful in most cases, because the virus load was below the detection limit or the virus was in its latent stage. Therefore, detection of antibodies against selected recombinant proteins corresponding to epitopes detected by nearly all infected animals in a Western blot assay is advantageous. By contrast, immunological testing is not beneficial in young animals as piglets might have PCMV/PRV-specific antibodies obtained from their infected mother via the colostrum. Using a thoughtful combination of PCR-based and immunological methods, detection of PCMV/PRV in donor pigs for xenotransplantation is feasible and a controlled elimination of the virus by early weaning or other methods is possible

Quantifying metabolic activity of Ascaris suum L3 using resazurin reduction

Background Helminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine. Methods Ascaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity. Results We show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100–1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO2 for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively. Conclusions Together, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro

Toward a porcinein vivomodel to analyze the pathogenesis of TLR5-dependent enteropathies

Non-communicable diseases, such as the metabolic syndrome and inflammatory bowel disease, constitute serious public health threats in developed countries. Besides environmental factors, genetic predispositions contribute to the onset and progression of the disease.State-of-the-artmouse models recently highlight the involvement of Toll-like receptor 5 (TLR5)-driven microbiota composition in the development of metabolic disorders. Here, we discuss the causes and consequences of an altered enteric microbiota and provide information on a similar mechanism in another species, the pig. We show for the first time that a single nucleotide polymorphism in the porcineTLR5gene conferring impaired functionality is associated with changes in the intestinal microbiota in adult sows and neonatal piglets. Changes in the developing adaptive cellular immune response support the concept of TLR5-driven changes of the microbe-host interplay also in the pig. Together, these findings suggest that pigs with impaired TLR-functionality might represent a model for TLR5-driven diseases in humans

The Worm-Specific Immune Response in Multiple Sclerosis Patients Receiving Controlled Trichuris suis Ova Immunotherapy

Considering their potent immunomodulatory properties, therapeutic applications of Trichuris suis ova (TSO) are studied as potential alternative treatment of autoimmune disorders like multiple sclerosis (MS), rheumatoid arthritis (RA), or inflammatory bowel disease (IBD). Clinical phase 1 and 2 studies have demonstrated TSO treatment to be safe and well tolerated in MS patients, however, they reported only modest clinical efficacy. We therefore addressed the cellular and humoral immune responses directed against parasite antigens in individual MS patients receiving controlled TSO treatment (2500 TSO p.o. every 2 weeks for 12 month). Peripheral blood mononuclear cells (PBMC) of MS patients treated with TSO (n = 5) or placebo (n = 6) were analyzed. A continuous increase of serum IgG and IgE antibodies specific for T. suis excretory/secretory antigens was observed up to 12 months post-treatment. This was consistent with mass cytometry analysis identifying an increase of activated HLA-DRhigh plasmablast frequencies in TSO-treated patients. While stable and comparable frequencies of total CD4+ and CD8+ T cells were detected in placebo and TSO-treated patients over time, we observed an increase of activated HLA-DR+CD4+ T cells in TSO-treated patients only. Frequencies of Gata3+ Th2 cells and Th1/Th2 ratios remained stable during TSO treatment, while Foxp3+ Treg frequencies varied greatly between individuals. Using a T. suis antigen-specific T cell expansion assay, we also detected patient-to-patient variation of antigen-specific T cell recall responses and cytokine production. In summary, MS patients receiving TSO treatment established a T. suis-specific T- and B-cell response, however, with varying degrees of T cell responses and cellular functionality across individuals, which might account for the overall miscellaneous clinical efficacy in the studied patients