Christian Menge

Crossing the Interspecies Barrier: Opening the Door to Zoonotic Pathogens

This research was funded by EU FP7 grant ANTIGONE (#278976). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Bovine Immune Response to Shiga-Toxigenic Escherichia coli O157:H7

ABSTRACT Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx−E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx−O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+O157 was significantly higher than that of Stx−O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+O157. Following the challenge, levels of fecal shedding of Stx2+O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx−O157, but not those inoculated with Stx2+O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.

Bovine Ileal Intraepithelial Lymphocytes Represent Target Cells for Shiga Toxin 1 from Escherichia coli

ABSTRACT The discovery that bovine peripheral lymphocytes are sensitive to Stx1 identified a possible mechanism for the persistence of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) in the bovine reservoir host. If intraepithelial lymphocytes (IEL) are also sensitive to Stx1, the idea that Stx1 affects inflammation in the bovine intestine is highly attractive. To prove this hypothesis, ileal IEL (iIEL) were prepared from adult cattle, characterized by flow cytometry, and subjected to functional assays in the presence and absence of purified Stx1. We found that 14.9% of all iIEL expressed Gb3/CD77, the Stx1 receptor on bovine lymphocytes, and 7.9% were able to bind the recombinant B subunit of Stx1. The majority of Gb3/CD77+ cells were activated CD3+ CD6+ CD8α+ T cells, whereas only some CD4+ T cells and B cells expressed Gb3/CD77. However, Stx1 blocked the mitogen-induced transformation to enlarged blast cells within all subpopulations to a similar extent and significantly reduced the percentage of Gb3/CD77+ cells. Although Stx1 did not affect the natural killer cell activity of iIEL, the toxin accelerated the synthesis of interleukin-4 (IL-4) mRNA and reduced the amount of IL-8 mRNA in bovine iIEL cultures. Because the intestinal system comprises a rich network of interactions between different types of cells and any dysfunction may influence the course of intestinal infections, this demonstration that Stx1 can target bovine IEL may be highly relevant for our understanding of the interplay between STEC and its reservoir host.

Detection of colostrum-derived alloantibodies in calves with bovine neonatal pancytopenia.

Bovine neonatal pancytopenia (BNP) is an emerging calf disease of unknown cause characterized by a pronounced susceptibility to bleeding as a result of a pancytopenia and bone marrow depletion. In this study we investigated whether this phenomenon is related to colostrum-derived alloantibodies directed against neonatal leukocytes. In a first experiment and using a flow cytometric approach sera from 6 BNP-dams (had given birth to BNP-calves; vaccinated against bovine viral diarrhea virus [BVDV]) and 6 control-dams (no herd history of BNP; no BVDV vaccination) were analyzed for the presences of alloantibodies (IgG) able to bind to the surface of leukocytes isolated from 7 calves from a herd with no history of BNP (no BVDV vaccination). In a second experiment, 4 neonates from 3 BNP-dams were fed colostrum from their corresponding mothers and sampled on a regular basis from birth up to day 21 of life under clinically controlled conditions. Sample analysis of the 4 neonates included hematology (white blood cell count and platelets), bone marrow cytology and histopathology as well as the flow cytometric detection of the percentage of IgG+-lymphocytes/monocytes in the peripheral blood. Experiment #1 showed that all BNP-dam sera harbored significantly higher alloantibody titers than the control dam sera (p<0.001). In the peripheral blood of the two neonates (Experiment #2), the percentage of IgG+-cells increased dramatically within 12h post colostrum intake (p.c.i.), remaining at over 95% for up to 3 days. Both calves developed BNP-associated clinical symptoms, one died. Both twin calves showed no clinical symptoms accompanied by a minor increase of IgG+ cells for up to 12h. Thus, the level of IgG+-cells and the duration of the detection thereof correlated with the severity of BNP developed by these animals. The results show that BNP-dams harbor alloantibodies against surface antigens of neonatal leukocytes in their sera that are readily transferred to the offspring via colostrum. These alloantibodies probably play a crucial role in the pathogenesis of BNP.

Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1

ABSTRACT Bovine colonic crypt cells express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization of cattle by human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used flow cytometric and real-time PCR analyses of primary cultures of colonic crypt cells to evaluate cell viability, CD77 expression, and gene transcription in the presence and absence of purified Stx1. A subset of cultured epithelial cells had Stx receptors which were located mainly intracellularly, with a perinuclear distribution, and were resistant to Stx1-induced apoptosis and Stx1 effects on chemokine expression patterns. In contrast, a population of vimentin-positive cells, i.e., mesenchymal/nonepithelial cells that had high numbers of Stx receptors on their surface, was depleted from the cultures by Stx1. In situ, CD77+ cells were located in the lamina propria of the bovine colon by using immunofluorescence staining. A newly established vimentin-positive crypt cell line with high CD77 expression resisted the cytolethal effect of Stx1 but responded to Stx1 with a significant increase in interleukin-8 (IL-8), GRO-α, MCP-1, and RANTES mRNA. Combined stimulation with lipopolysaccharide and Stx1 increased IL-10 mRNA. Our results show that bovine colonic crypt cells of epithelial origin are resistant to both the cytotoxic and modulatory effects of Stx1. In contrast, some mucosal mesenchymal cells, preliminarily characterized as mucosal macrophages, are Stx1-responsive cells that may participate in the interaction of STEC with the bovine intestinal mucosa.

Verotoxin 1 from Escherichia coli affects Gb3/CD77+ bovine lymphocytes independent of interleukin-2, tumor necrosis factor-α, and interferon-α

Verotoxin (VT)-induced immunomodulation has been implicated in the ability of VT-producing Escherichia coli (VTEC) to cause persistent infections in cattle. VT1, also referred to as Shiga toxin 1, is a potent cytotoxin that modulates cytokine secretions and functions. This prompted the current investigation to examine whether the inhibiting effect of VT1 on bovine lymphocytes correlates with the expression of the cellular VT1 receptor Gb3/CD77 or is mediated instead via perturbation of cytokine secretion. Using blood mononuclear cells stimulated by mitogens as a model, VT1 significantly blocked lymphoblast transformation and proliferation in the BoCD8+ T cell and BoCD21+ B cell population. In contrast, VT1 dramatically reduced the number of viable Gb3/CDTZ+ blast cells within all subpopulations identified (BoCD2+, BoCD4+, BoCD8+, WC1+ [i.e., γδ T cells] BoCD21+, and BoCD25+). Similar effects of VT1 were observed when the culture medium was supplemented with selected cytokines: tumor necrosis factor-α-sensitizing endothelial cells against VT1, interferon-a (IFN-α) as bovine IFN-α receptors are partially homologous to the B-subunit of VT1, and interleukin-2 that is critical for lymphocyte proliferation in vitro. The addition of these cytokines was neither able to mimic nor to overcome the effects of VT1. Therefore, it is concluded that VT1 directly acts on bovine lymphocytes rather than inducing a cytokine-mediated effect. VT1 considerably affects all main bovine lymphocyte subpopulations, implicating that the immune system is a predominant target for VT1 In cattle.

Globotriaosylceramide (Gb3/CD77) is synthesized and surface expressed by bovine lymphocytes upon activation in vitro

Neutral glycosphingolipids (GSLs) are considered activation markers on human lymphocytes, which are fundamental for studying the immune system. For cattle, only a limited number of activation markers has yet been identified. We recently showed that Shiga toxin 1, known to use globotriaosylceramide (Gb(3) syn. CD77) as a cellular receptor, depresses proliferation of activated bovine lymphocytes [Infect. Immunol. 67 (1999b) 2209]. In order to confirm the expression of Gb(3)/CD77 on bovine lymphocytes, we flowcytometrically examined a bovine B-lymphoma cell line (BL-3) and bovine peripheral blood mononuclear cells (PBMC) before and after mitogenic stimulation and biochemically characterized neutral GSLs extracted from PBMC. CD77 was detected on the surface of BL-3 cells and cultured PBMC essentially after mitogenic stimulation. Although expressed by all PBMC subpopulations identified, the portion of CD7+ cells was highest for BoCD8+ cells, followed by B-cells and BoCD4+ cells at day 4 of cultivation. Ceramide trihexoside of stimulated PBMC was structurally determined as Gal(alpha1-4)Gal(1-4)Glc(1-1)ceramide (Gb(3)). Biochemically, Gb(3) was also detected within unstimulated PBMC which contained ceramide monohexoside (CMH) and Gb(3) in a ratio of about 4:1. However, stimulation induced an increase of CMH and Gb(3) by a factor of 2.5 and 10, respectively, implicating that bovine lymphocytes regulate surface expression of Gb(3)/CD77 predominantly by quantitative changes in the Gb(3) metabolism. This report presents Gb(3)/CD77 as the first GSL identified on bovine immune cells and highly recommends this activation dependent antigen as a useful tool to investigate lymphocyte activation within the bovine immune system.

Primary bovine colonic cells: a model to study strain-specific responses to Escherichia coli.

The parasitic or commensal lifestyle of bacteria in different hosts depends on specific molecular interactions with the respective host species. In vitro models to study intestinal bacteria-host interactions in cattle are not available. Bovine primary colonocyte (PC) cultures were generated from colon crypt explants. Up to day 4 of culture, the vast majority of cells were of epithelial phenotype (i.e., expressed cytokeratin but not vimentin). PCs harboured mRNA specific for Toll-like receptors (TLR) 1, TLR3, TLR4 and TLR6 but not for TLR2, TLR5, TLR7, TLR8, TLR9 and TLR10. Six hours after inoculation of PC cultures with Escherichia coli (E. coli) prototype strains representing different pathovars (enterohaemorrhagic E. coli [EHEC], enteropathogenic E. coli [EPEC], enterotoxic E. coli [ETEC]), bacteria were found attached to the cells. EPEC adhesion was accompanied by intracellular actin accumulation. An attenuated laboratory strain (E. coli K12 C600) and a bovine commensal E. coli strain (P391) both did not adhere. Bacterial or LPS challenge of PC cultures resulted in specific increases in mRNA transcripts for IL-8, GRO-alpha, MCP-1, RANTES, and IL-10. The level of mRNA transcripts for TGF-beta stayed constant, while IL-12 mRNA was not detectable. Short-term cultures of PCs, maintaining epithelial cell properties, interacted with commensal and pathogenic bacteria in a strain-specific manner and have proven to be a useful in vitro model to study the interaction of bacteria with the bovine intestinal mucosa.

Bovine Caruncular Epithelial Cell Line (BCEC-1) Isolated from the Placenta Forms a Functional Epithelial Barrier in a Polarised Cell Culture Model

Abstract In the bovine synepitheliochorial placenta key sites of fetal–maternal interaction are placentomes consisting of maternal caruncles interdigitating with fetal cotyledons. The aim of this study was to establish an epithelial cell line from caruncles of pregnant cows and to develop a model to study restricted trophoblast invasion, pathogenesis of pregnancy associated diseases and pathways of infection and transport. Primary epithelial cells were isolated, successfully subcultured for 32 passages and cryopreserved at various stages. The cultures were termed bovine caruncular epithelial cell line-1 (BCEC-1). Cytokeratin, zonula occludens-1 protein and vimentin but neither α-smooth muscle actin nor desmin were detected by immunofluorescence performed every 5 (±1) passages. These results were confirmed by Western blotting. BCEC-1 were then cultured either without matrix or on fibronectin or collagen coated Transwell® polyester membrane inserts, respectively, enabling separate access to the basal or apical epithelial compartments. Transmission and scanning electron microscopy of BCEC-1 revealed ultrastructural features also observed in vivo, such as apical microvilli and junctional complexes. Transepithelial electrical resistance (TEER) was measured regularly and revealed an increase with advancing confluence in all cultures. Cultures on coated inserts reached confluence and corresponding TEER-levels at an earlier stage. In addition, the cells were tested negative for bovine virus diarrhoea (BVD) virus, but were permissive for the virus. In conclusion, the BCEC-1 cell line retained characteristics of maternal caruncular epithelial cells as observed in vivo and in primary cell cultures and thus will be a highly useful tool for future studies of pathways of invasion, fetal–maternal communication, transport and infection.

Distinct Intensity of Host-Pathogen Interactions in Chlamydia psittaci- and Chlamydia abortus-Infected Chicken Embryos

ABSTRACT Factors and mechanisms determining the differences in virulence and host specificity between the zoonotic agents Chlamydia psittaci and Chlamydia abortus are still largely unknown. In the present study, two strains were compared for their invasiveness, virulence, and capability of eliciting an immune response in chicken embryos. On breeding day 10, embryonated chicken eggs were inoculated with 5 × 104 inclusion-forming units. As shown by immunohistochemistry and quantitative real-time PCR, C. psittaci displayed a significantly better capability of disseminating in the chorioallantoic membrane (CAM) and internal organs than C. abortus. The higher infectious potential of C. psittaci in birds was underlined by significantly higher mRNA expression rates of essential chlamydial genes, such as incA, groEL (in CAM, liver, and spleen), cpaf, and ftsW (in CAM). Although the immune responses to both pathogens were similar, C. psittaci elicited higher macrophage numbers and a stronger expression of a subset of immune-related proteins. The data imply that invasiveness of Chlamydia spp. and propagation in the host are not solely dependent on the level of host immune response but, even to a greater extent, on the expression of bacterial factors related to virulence. The fact that C. psittaci has coped far better than C. abortus with the avian embryos response by upregulating essential genes may be a key to understanding the mechanisms underlying host adaptation and etiopathology.