Based on epidemiological and clinical observations, different strains of Mycobacterium avium subsp. paratuberculosis (MAP) are suspected to significantly differ in their virulence for ruminants. In the pathogenesis of paratuberculosis, macrophages represent the principal target cell for MAP. In order to judge the ability of different MAP-genotypes to modulate macrophage responses, the cytokine responses of the monocyte cell line THP-1 were studied after challenge with three different MAP strains under standardized conditions. The bovine field isolate J1961 (major Type II) and the ovine field isolate JIII-86 (Type III) were compared with the laboratory adapted reference strain ATCC 19698 (Type II). Strains were shown by three different typing methods (IS900-RFLP-, MIRU-VNTR-, and SSR-analysis) to substantially differ in several genotypic features. Macrophage function was assessed by quantifying mRNA of the cytokines TNF-α, IL-1ß, and IL-10 by quantitative RT-PCR. Secreted TNF-α protein was measured by a cytotoxicity test, IL-1ß and IL-10 using ELISA tests. The three MAP strains of various genotypes differ in their effect on human macrophages depending on challenge dose and infection time. These differences concerned both the mRNA level and secreted protein amounts of proinflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokine IL-10. Type III strain produced less IL-10 and IL-1β mRNA and protein but more TNF-α protein at 2h than the Type II strains. In summary, our results support the hypothesis that strain characteristics might have relevance for the host response towards MAP and, consequently, for the pathogenesis of paratuberculosis.

ABSTRACT eibG in Shiga toxin-producing Escherichia coli (STEC) O91 encodes a protein (EibG) which binds human immunoglobulins G and A and contributes to bacterial chain-like adherence to human epithelial cells. We investigated the prevalence of eibG among STEC, the phylogeny of eibG , and eibG allelic variations and their impact on the adherence phenotype. eibG was found in 15.0% of 240 eae -negative STEC strains but in none of 157 eae -positive STEC strains. The 36 eibG -positive STEC strains belonged to 14 serotypes and to eight multilocus sequence types (STs), with serotype O91:H14/H − and ST33 being the most common. Sequences of the complete eibG gene (1,527 bp in size) from eibG -positive STEC resulted in 21 different alleles with 88.11% to 100% identity to the previously reported eibG sequence; they clustered into three eibG subtypes ( eibG -α, eibG -β, and eibG -γ). Strains expressing EibG-α and EibG-β displayed a mostly typical chain-like adherence pattern (CLAP), with formation of long chains on both human and bovine intestinal epithelial cells, whereas strains with EibG-γ adhered in short chains, a pattern we termed atypical CLAP. The same adherence phenotypes were displayed by E. coli BL21(DE3) clones containing the respective eibG -α, eibG -β, and eibG- γ subtypes. We propose two possible evolutionary scenarios for eibG in STEC: a clonal development of eibG in strains with the same phylogenetic background or horizontal transfer of eibG between phylogenetically unrelated STEC strains.

The ability to colonize the chicken gut was determined for 17 Campylobacter jejuni strains of human and bovine origin. The level of colonization varied according to the strain used for experimental infection. Two Campylobacter isolates from patients suffering from gastroenteritis were found in the group of non-colonizing strains, suggesting that other reservoirs as poultry are also important sources of human Campylobacter infections. Bovine Campylobacter isolates can also effective colonize the chicken intestine and may be a source for poultry infection. The invasion ability of the strains as determined in the cell culture model using Caco-2 cells correlates with their colonization capacity in the chicken gut. The genomic and phenotypic stability of the selected strains were evaluated by analysis of their pulsed-field gel electrophoresis (PFGE) patterns, flaA-typing and in vitro determination of motility, adhesion and invasion abilities after colonizing chickens for up to 21 days. Changes were identified in flaA-types of six isolates and three isolates from chicken showed different patterns by PFGE using SmaI or KpnI as restriction enzymes. One isolate showed phenotypic differences after in vivo passage which were seen in enhancement of adherence to eukaryotic cells, decrease of motility and changes in morphology. These phenotypic changes were not associated with the observed genomic instabilities.

Campylobacter (C.) jejuni and C. coli can cause gastrointestinal disorders in humans characterized by acute inflammation. Inflammatory signals are initiated during interaction between these pathogens and human intestinal cells, but nothing is known about the stimulation of avian intestinal cells by Campylobacter. Interleukin-8 (IL-8) as a proinflammatory chemokine plays an important role in mobilizing cellular defence mechanism. IL-8 mRNA expression in both human intestinal cells (INT 407) and primary intestinal chick cells (PIC) was determined by quantitative real-time RT-PCR. The secretion of IL-8 protein by INT407 was measured using ELISA. Although C. jejuni and C. coli are considered to be harmless commensals in the gut of birds, the avian Campylobacter isolates investigated were able to induce the proinflammatory IL-8 in PIC as well as in INT407. In an in vitro system, C. jejuni as well as C. coli were able to induce IL-8 mRNA in PIC. Relation between the virulence properties like toxin production, the ability to invade and to survive in Caco-2 cells and the level of IL-8 mRNA produced by INT 407 and PIC after infection with Campylobacter strains was also investigated.

Cell culture assays are possible alternatives to replace in vivo neutralization tests currently required for potency testing of clostridial vaccines. Cell culture assays based on the MDCK cell line and the Vero cell line which are sensitive to the Clostridium (C.) perfringens type D epsilon toxin and Clostridium novyi type B alpha toxin, respectively, were developed, and the test conditions were standardized. The antibody titres of vaccinated rabbits measured in vitro were compared with the results of current test procedures recommended by European Pharmacopoeia. The correlation coefficients calculated were significant for all sera tested. The cell culture assays proved to be sensitive, specific, reproducible and reliable. Therefore, these cell culture assays could be suitable in vitro alternatives to the in vivo mouse neutralization experiments required for potency tests of clostridial vaccines, but further validation studies are necessary.

Campylobacter isolates from turkeys were genotyped and characterized by their in vitro virulence properties. Relationships between bacterial genotypes and virulence properties were analysed.Isolates were analysed by pulsed-field gel electrophoresis and fla typing. The toxin production was determined on the phenotypic level using a CHO-K1 cell culture model and on the genotypic level using PCR for detection of the cdtA, cdtB and cdtC genes. Although the cdtB gene was detected from 100% of the Campylobacter jejuni and Campylobacter coli isolates we observed three different morphological pictures on the cells. Cytotoxicity was associated with cell distension or cell rounding. All four Camp. coli strains and one Camp. jejuni strain did not produce any cytotoxic changes on the cells. Adhesion, invasion and survival of Campylobacter isolates were determined in a Caco-2 cell culture model. All isolates adhered to and invaded Caco-2 cells, whereas 64.7% of the strains survived for 48 h in the cells.Seventeen Campylobacter isolates from turkeys were classified into four groups with regard to their in vitro abilities. Jackknife analysis revealed a strong association between these groups and genotype clusters.Typing methods have generally failed to identify strains with specific virulence properties. This study suggests that a relationship between subgroups of Campylobacter with common in vitro virulence characteristics and genotypes exist.

The toxin binding inhibition test (ToBI) were developed for potency testing of C. novyi type B alpha toxoid containing veterinary vaccines to replace the currently used toxin neutralisation test in mice (TNT). The antitoxin titres of rabbit sera (AN-, HV- and SP sera) were determined with ToBI using the international reference serum with known antitoxin titre. In order to show the validity of the methods, the results were compared with those of the manufacturers/regulatory authorities and correlation coefficients were calculated. The correlation coefficients were r = 0.93 (AN sera), r = 0.73 (HV sera) and r = 0.85 (SP sera). All correlations were statistically significant. The specificity of the methods could be proved using heterologous antisera. The results of the ToBI were reproducible. Thus, the ToBI offers a suitable in vitro method for the determination of the antitoxin titre of rabbit antisera as an alternative to the toxin neutralisation in mice for potency testing of vaccines containing C. novyi type B alpha toxoid.

Animal experiments still play a central role in the quality control of vaccines. Generally, performance of these experiments is provided by law and laid down in the European Pharmacopoeia. Classical vaccines, the efficacy of which is calculated in International Units, require a very high number of experimental animals for quality control testing. The testing mainly consists of infection and intoxication experiments causing extreme suffering of the animals involved. This classical product group includes clostridial vaccines which are used to a great extent in veterinary medicine in particular. Within the last years, considerable efforts have been made to reduce the number of animal experiments in this field, lower the number of animals, and decrease the suffering of the animals during testing. Several research projects for the development and validation of alternative methods have been initiated. Furthermore, the 3R Concept (Replacement, Reduction and Refinement) is increasingly taken into consideration when developing or revising legal provisions. This led to various improvements regarding animal welfare in the quality control of clostridial vaccines.

Cell culture assays, using the MDCK cell line was confirmed as being sensitive to the C. perfringens epsilon toxin and VERO cell line to the C. novyi type B alpha toxin. Cell culture assays using these cells were developed and the test conditions were standardised. The antitoxin titres of rabbit antisera were calculated anc compared with those of the manufacturers. The correlation coefficients between in vitro and in vivo method were calculated and were significant.The cell culture assay offers a valid in vitro alternative tot the animal experiments for the titration of sera generated in the course of potency tests of clostridial vaccines.

Epsilon toxin is one of the major lethal toxins produced by Clostridium perfringens type D and B. It is responsible for a rapidly fatal disease in sheep and other farm animals. Many facts have been published about the physical properties and the biological activities of the toxin, but the molecular mechanism of the action inside the cells remains unclear. We have found that the C. perfringens epsilon toxin caused a significant decrease of the cell numbers and a significant enlargement of the mean cell volume of MDCK cells. The flow cytometric analysis of DNA content revealed the elongation of the S phase and to a smaller extent of the G2+M phase of toxin-treated MDCK cells in comparison to untreated MDCK cells. The results of ultrastructural studies showed that the mitosis is disturbed and blocked at a very early stage, and confirmed the toxin influence on the cell cycle of MDCK cells.

The aim of our study was to investigate if a cell culture assay can replace the toxin neutralisation test in mice for the potency testing of alpha toxoid containing clostridial vaccines for veterinary use. The basis for the development of our cell culture assay was the detection of the cytotoxic/cytopathic action of alpha toxin on cells in culture. Nine permanent cell lines were examined for their reaction to the alpha toxin. The action of the toxin was determined after three days by microscopic examination and MTT assay. The alpha toxin exhibited the strongest effect on the ESH-L cells. We were able to show that the cytopathic effect was neutralised by the international standard for gas gangrene antitoxin (C.novyi) but never by heterologous antisera. Our results showed that the ESH-L cell line was a suitable indicator for the detection of the cytotoxic effect of alpha toxin.