PubMed Health⌕ Search

Biomedical subjects

Georg Baljer

Publications and source records attributed to Georg Baljer.

14 recordsLinked to original sources
PubMed2006

Escherichia coli Shiga toxin 1 enhances il-4 transcripts in bovine ileal intraepithelial lymphocytes.

Shiga toxin 1 (Stx1) blocks the activation of bovine peripheral and intraepithelial lymphocytes (IEL), implying that the toxin has the potential to retard the host's immune response during intestinal colonization of cattle with human pathogenic Stx-producing Escherichia coli (STEC). Since Stx1 does not eliminate affected lymphocytes by causing cellular death, we assumed that Stx1 disturbs the integrity of the immune regulatory network. We therefore assessed the impact of Stx1 on the expression of selected chemokine and cytokine genes in vitro by real-time RT-PCR and by quantitation of intracellular cytokine proteins. While Stx1 did not alter the amount of mRNA specific for interleukin (IL)-2, IL-10, gamma interferon (IFN-gamma), transforming growth factor beta (TGF-beta), IL-8, 10kDa interferon inducible protein (IP-10), and monocyte chemoattractant protein 1 (MCP-1) in cultured ileal IEL (iIEL), minute concentrations of Stx1 led to an up to 40-fold increase of il-4 transcripts within 6-8h of incubation. Comparative experiments with peripheral lymphocytes revealed that the effect was specific for iIEL. The enhancement of il-4 transcripts in iIEL was not accompanied by apoptosis but required the enzymatic activity of the holotoxin. Nevertheless, iIEL retained their ability to synthesize proteins in the presence of Stx1: 40% of iIEL could be stimulated to synthesize IFN-gamma while less than 10% expressed IL-4 or TGF-beta. Furthermore, iIEL were found to produce granulocyte chemoattractants, but the release of these substances was not different in iIEL cultures incubated with or without Stx1. Although Stx1 did not affect the numbers of iIEL producing either cytokine, these findings point to an altered responsiveness of IEL during bovine STEC infections and shed light on the initial effects Stx1 exerts on the local adaptive immune system.

Animals↗
PubMed2006

Comparison of binding and effects of Escherichia coli Shiga toxin 1 on bovine and ovine granulocytes.

Granulocytes play a pivotal role in the pathogenesis of Shiga toxin (Stx)-producing Escherichia coli (STEC) related diseases in humans. Granulocytes are attracted and activated by Stxs in the enteric mucosa and are believed to thereby contribute to the intestinal inflammation. Mature ruminants, the main reservoir hosts of STEC, do not develop pathological changes that can be attributed to the Stxs. To prove whether the latter phenomenon correlates with the inability of the Stxs to affect granulocytes of ruminants, we investigated the ability of Stx1 to bind to granulocytes of cattle and sheep and analysed the effects of Stx1 on viability, phagocytosis, and oxidative burst activity. Bovine granulocytes from blood and milk did not express Stx1-binding sites even after activation of the cells and also were resistant to Stx1. In contrast to bovine granulocytes, granulocytes of sheep constitutively expressed Stx1-receptors of the Gb(3)/CD77 type ex vivo and bound the recombinant B-subunit of Stx1 (rStxB1). Stx1 holotoxin induced apoptosis in ovine granulocytes after prolonged incubation (18h) but Stx1 only slightly altered the phagocytosis and oxidative burst activities. The rStxB1 had no effect on granulocytes of either species. While arguing in favour of our initial hypothesis, that granulocytes of both, cattle and sheep are not activated by Stxs, the results of our study are the first evidences for differences in the cellular distribution of Stx-receptors in species equally regarded as STEC carriers.

Animals↗
PubMed2006

Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii.

BACKGROUND: Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. RESULTS: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 10(7) starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. CONCLUSION: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.

Coxiella burnetii↗
PubMed2005

Immunization with an alphatoxin variant 121A/91-R212H protects mice against Clostridium perfringens alphatoxin.

As shown previously, a recombinant alphatoxin variant (rAT121A/91) constructed from the naturally occurring Clostridium perfringens mutant strain 121A/91, was devoid of enzymatic (PLC), hemolytic and lethal activity (18). In the present study, the recombinant variant was altered by an oligonucleotide-directed reversion of an arginine in position 212 for a histidine residue, corresponding to the sequence of the wild-type alphatoxin. The new variant rAT121A/91R212H proved to be negative in enzymatic, hemolytic and lethal activity as well. RAT121A/91 as well as rAT121A/91R212H was used for i.p. immunization of balb/c mice. The immune response was studied in ELISA as well as in the mouse neutralization test. Furthermore, immunized mice were challenged by i.p. application of active C. perfringens alphatoxin. In all immunized groups, mice developed high anti-alphatoxin titers (up to 1:128000). Antisera of both groups were able to reduce the hemolytic effect of native alphatoxin with predominance of anti-rAT121A/91R212H sera. During neutralization experiments, mice receiving a mixture of anti-rAT121A/91R212H and wild-type toxin were protected completely, whereas an anti-rAT121A/91/toxin mixture prolonged time until death but failed in protection. I.p immunization with rAT121A/91R212H yielded a significant protection rate (76%) when mice were challenged intraperitoneal with wild-type toxin. Our cumulative data indicates that the reversion of arginine in position 212 to histidine for rAT121A/91R212H was necessary to induce production of protective antibodies against wild-type alphatoxin of C. perfringens.

Animals↗
PubMed2005

Immunization experiments with recombinant Coxiella burnetii proteins in a murine infection model.

Previous attempts to develop Q fever vaccines were less successful in that the vaccines caused unacceptable side effects or failed to be protective. In this study, we tested the efficacy of a mixture of eight recombinant Coxiella burnetii (C. b.) proteins in sublethal challenge infections with mice. Eight potential C. b. virulence genes (Omp, Pmm, HspB, Fbp, Orf410, Crc, CbMip, and MucZ) were overexpressed in E. coli as his-tagged fusion proteins and partially purified. All recombinant proteins but rPmm proved to be antigenic in BALB/c mice when administered as protein mixtures. For efficacy testing, mice were immunized with an adjuvanted mixture of the eight recombinant proteins and subsequently challenged intraperitoneally with the C. b. isolate Nine Mile RSA493 (1.8 x 10(8) C. b.). Only animals vaccinated with the licensed Q fever vaccine Q-Vax (vaccination control) exhibited milder symptoms and minor gain of spleen and liver weights. In summary, clinical examinations and dissection of mice immunized with the eight recombinant C. b. proteins did not indicate a protective immune response after test infection.

Animals↗
PubMed2004

Molecular characteristics of Escherichia coli serogroup O78 strains isolated from diarrheal cases in bovines urge further investigations on their zoonotic potential.

We investigated the virulence properties and clonal relationship of 21 Escherichia coli strains of serogroup O78 isolated from diarrhoeic cattle and calves. Isolates were screened for 18 genes representing virulence features of different Escherichia coli pathotypes. None of the strains harboured enterotoxin-genes estIa/Ib, eltIa/Ib, or Shiga toxin (stx) genes, genes involved in adhesion (eae, f5, f41) hemolysin gene hlyA or invasion gene ipaC. With a high prevalence we detected enterotoxin astA (61.9%), genes involved in iron acquisition, like fyuA, irp (each 57.1%) and iucD (81.0%), and the operon sequence of Colicin V plasmids (38.1%). Some strains possessed toxin genes cdt-IIIB and cnf1/2 (both 14.3%), the invasion gene tia (23.8%), and the serine protease encoding gene espP (23.8%). Moreover, we could show that E. coli O78 strains under investigation were able to adhere to and invade MDBK-cells with varying efficiencies. The results indicate that the closely related O78 strains, constituting two major PFGE-clusters, harbor various virulence features for bovine intestinal disease but cannot be grouped into one of the common E. coli intestinal pathogenic or other pathotypes according to their virulence gene pattern. Nevertheless, the ability to adhere, invade or harbor toxin genes lets us suggest that O78 strains isolated from diarrheal cases in bovines urges further investigations on the zoonotic potential of these strains.

Animals↗
PubMed2004

Phenotypic and functional characterization of intraepithelial lymphocytes in a bovine ligated intestinal loop model of enterohaemorrhagic Escherichia coli infection.

Ruminants are a major reservoir of enterohaemorrhagic Escherichia coli (EHEC), which cause acute gastroenteritis in humans with potentially life-threatening sequelae. The mechanisms underlying EHEC persistence in ruminant hosts are poorly understood. EHEC produce several cytotoxins that inhibit the proliferation of bovine lymphocytes in vitro and influence EHEC persistence in calves, suggesting that bacterial suppression of mucosal inflammation may be important in vivo. In order to address this hypothesis, intraepithelial lymphocytes (IEL) obtained from ligated intestinal loops of five 9-14 day old calves were characterized 12 h after inoculation with E. coli strains. Loops were inoculated with an EHEC O103 : H2 strain, an isogenic Deltastx1 mutant incapable of producing Shiga toxin 1 (Stx1) and a porcine non-pathogenic E. coli strain. The IEL mainly comprised activated CD2(+) CD3(+) CD6(+) CD8alpha(+) T cells and resembled IEL obtained from the intestinal mucosa of orally challenged calves. Forty per cent of all IEL were potentially sensitive to Stx1 in that they expressed the receptor for Stx1. Nevertheless, analysis of IEL from inoculated loops failed to detect a significant effect of the different E. coli strains on proliferative capacity, natural killer cell activity or the cytokine mRNA profile. However, the EHEC wild-type strain reduced the percentage of CD8alpha(+) T cells in the ileal mucosa compared with loops inoculated with the Deltastx1 mutant. This shift in IEL composition was not associated with inhibition of IEL proliferation in situ, since the majority of the IEL from all loops were in the G(0)/G(1) phase of the cell cycle. These studies indicate that the ligated ileal loop model will be a useful tool to dissect the mechanisms underlying suppression of mucosal inflammation by EHEC in the reservoir host.

Animals↗
PubMed2004

Bovine ileal intraepithelial lymphocytes represent target cells for Shiga toxin 1 from Escherichia coli.

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 Gb(3)/CD77, the Stx1 receptor on bovine lymphocytes, and 7.9% were able to bind the recombinant B subunit of Stx1. The majority of Gb(3)/CD77(+) cells were activated CD3(+) CD6(+) CD8 alpha(+) T cells, whereas only some CD4(+) T cells and B cells expressed Gb(3)/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 Gb(3)/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.

Animals↗
PubMed2003

Differences in cytokine mRNA profiles between naïve and in vivo-primed ovine PBMC after exposure to heat-inactivated Coxiella burnetii.

During human Coxiella burnetii (C. burnetii) infections, high IL-10 levels favor replication of C. burnetii in monocytes and development of chronic Q fever, whereas IFN-gamma promotes intracellular killing. Sheep are a common source for human C. burnetii infections, but in contrast to man become transiently infected only. In a first approach to unravel the role of cytokines during ovine C. burnetii infections, we investigated by semiquantitative RT-PCR whether heat-inactivated C. burnetii affects the transcription of genes coding for IL-2, IL-4, IL-10, and INF-gamma in vitro in PBMC from sheep seropositive or seronegative for C. burnetii. By computer-assisted evaluation of band intensities the transcription rate of the cytokine genes was quantified in relation to transcription in Concanavalin A-stimulated and nonstimulated controls. Transcription rates in PBMC from seropositive animals after incubation with C. burnetii for 4 hours strongly resembled those found in PBMC from seronegative sheep. However, upon prolonged incubation (24 h) C. burnetii induced an increased IL-10 transcription in PBMC from 2 of 5 seronegative, but in PBMC from 5 of 5 seropositive animals. The data suggest that natural C. burnetii infections prime the ovine immune system towards a T(H)2-like pattern and this action thereby represents the first clue for the involvement of ovine immune cells in the response to C. burnetii infections.

Animals↗
PubMed2003

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

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/CD77+ blast cells within all subpopulations identified (BoCD2+, BoCD4+, BoCD8+, WC1+ [i.e., gammadelta T cells] BoCD21+, and BoCD25+). Similar effects of VT1 were observed when the culture medium was supplemented with selected cytokines: tumor necrosis factor-alpha-sensitizing endothelial cells against VT1, interferon-alpha (IFN-alpha) as bovine IFN-alpha 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.

Animals↗
PubMed2002

Coxiella burnetii plasmid types QpDG and QpH1 are closely related and likely identical.

Strains and isolates of Coxiella burnetii, an obligate intracellular bacterium, carry a single plasmid or a plasmid-homologous sequence integrated into the chromosome. The plasmids QpH1, QpRS, QpDV and the chromosome-integrated plasmid-homologous region have been completely sequenced, whereas no sequence data are available for the QpDG plasmid. In this study, we used total genomic DNA from reference strain C. burnetii Dugway 5J108-111 to demonstrate and characterize the QpDG plasmid by pulsed-field gel electrophoresis (PFGE) and Southern hybridization. Primers derived from regions shared among C. burnetii plasmids were used to construct a physical map of the QpDG plasmid by extra long (XL) PCR. Both approaches, Southern hybridization and XL PCR indicated that QpDG and QpH1 represent a closely related and likely identical plasmid.

Animals↗
PubMedDate not supplied

[Distribution of Brachyspira hyodysenteriae and Lawsonia intracellularis in healthy and diarrhoeic pigs].

Although Brachyspira (B.) hyodysenteriae and Lawsonia (L.) intracellularis are widely distributed in pigs in Germany, there exists limited information on their clinical relevance. To get more insight into their potential role in swine diarrhoeal disease, in 2002 and 2003 faecal specimens from healthy pigs (n=1445) as well as from diarrhoeic pigs (n=2002) were tested by polymerase chain reaction (PCR) for the presence of both agents. Of the specimens from healthy pigs L. intracellularis and B. hyodysenteriae were detected in 7.3% and 6.7%, respectively. In contrast, of the diarrhoeic pigs the ratios of positive samples amounted to 19.4% for L. intracellularis and 17.9% for B. hyodysenteriae. Concerning the age of the diseased animals, in growing pigs the detection rates of L. intracellularis and B. hyodysenteriae were nearly identical (16.4% and 14.2%, respectively). In fattening pigs a significant higher number of animals were affected with B. hyodysenteriae (35.8%) than with L. intracellularis (28.2%). On the other hand, in sows L. intracellularis (35.6% positive samples) was dominant compared to B. hyodysenteriae (21.2% positive samples). Considering the nearly threefold higher percentage rates of L. intracellularis and B. hyodysenteriae in diarrhoeic pigs in comparison to healthy pigs, it is concluded that both agents play an important role in swine diarrhoeal disease. The results further indicated that in fattening pigs B. hyodysenteriae and in sows L. intracellularis have a dominant role, respectively.

Animals↗
PubMedDate not supplied

Racing, ornamental and city pigeons carry shiga toxin producing Escherichia coli (STEC) with different Shiga toxin subtypes, urging further analysis of their epidemiological role in the spread of STEC.

Pigeons are known to shed zoonotic pathogens. Therefore, in this study a total of 366 droppings from pigeons were analysed using PCR and DNA-DNA-hybridization for Shiga toxin producing E. coli (STEC). Specimens were collected from three different groups of pigeons: 247 collective and 3 individual droppings from racing pigeons, 26 collective and 40 individual from ornamental pigeons as well as 50 collective droppings from city pigeons. Initial screening experiments revealed a total 245 (66.9%) droppings to be Shiga toxin gene positive. Of these 36% were positive for stx1, 9% for stx2 and 37% for stx2f. Prevalence significantly (p < 0.001) differed in regard to the pigeon groups examined. Droppings from racing pigeons showed prevalence of 45.6% for stx1, 3.2% for stx2, and 33.2% for stx2f, while the distribution of stx-positive specimens was more even in ornamental pigeons (15% stx1, 27% stx2, and 26% stx2f). In specimens from city pigeons, stx2f was found to be most prevalent with 76% (2% stx1, 16% stx2). In 161 samples, stx genes were detected by PCR as well as DNA-DNA-hybridization. From these 161 samples, 20 were randomly chosen for isolation of STEC. A total of 27 STEC strains were isolated from 13 of these 20 samples. Six of the STEC were positive for stx1, 21 harbored stx2f. Further typing for virulence factor genes revealed the existence of eae in 4 of the 6 stx1-positive strains, as well as in 19 of the 21 stx2f-positive strains. eae is known to be crucially involved in the ability of E. coli strains to cause the "attaching and effacing" lesion in the gut, while stx2fSTEC are assumed to be host specific for pigeons. Here we report the first description of stx1- and eae-positive STEC strains in pigeons from Germany, especially in racing and ornamental pigeons. Taking into account the close contact between fanciers and pigeons, these findings warrant a more critical appraisal of these zoonotic pathogens in pigeons.

Animals↗