Genomic and phenotypic changes of Campylobacter jejuni strains after passage of the chicken gut.

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.

Relationships between bacterial genotypes and in vitro virulence properties of Campylobacter jejuni and Campylobacter coli isolated from turkeys.

AIMS: Campylobacter isolates from turkeys were genotyped and characterized by their in vitro virulence properties. Relationships between bacterial genotypes and virulence properties were analysed. METHODS AND RESULTS: 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. CONCLUSION: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Development of a cell culture assay for the quantitative determination of vaccination-induced antibodies in rabbit sera against Clostridium perfringens epsilon toxin and Clostridium novyi alpha toxin.

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.

[Progress in the replacement of animal experiments in the quality control of clostridial vaccines]. / Fortschritte beim Ersatz von Tierversuchen in der Qualitätskontrolle von Clostridien-Impfstoffen.

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.

[The use of the toxin binding inhibition test for potency testing of Clostridium novyi type B alpha toxoid vaccines]. / Anwendung des Toxin-Bindungs-Inhibitionstests zur Wirksamkeitsprüfung von Clostridium-novyi-Typ-B-Alphatoxoid-Impfstoffen.

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.

Effect of Clostridium perfringens epsilon toxin on MDCK cells.

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.

[Development of in vitro methods for the potency testing of clostridial vaccines]. / Entwicklung von in vitro Methoden zur Wirksamkeitsprüfung von Clostridien-Impfstoffen.

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 and 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 to the animal experiments for the titration of sera generated in the course of potency tests of clostridial vaccines.

Detection of Clostridium novyi type B alpha toxin by cell culture systems.

Ten permanent cell lines were examined for their reaction to the Clostridium novyi alpha toxin. The action of the toxin was determined after 3 days by microscopic examination and the MTT assay. The alpha toxin exhibited the strongest effect on ESH-L cells rather than other cell lines. Vero and SFT-R cells reacted in a comparable way, but less sensitively. We were able to show that the cytopathic effect on the three types of cells was neutralised by the international standard for gas gangrene antitoxin (C. novyi) but in no case by heterologous antisera. Our results have shown that the three cell lines were specific indicators for the detection of the cytopathic effect of alpha toxin. The cytopathic effect can be measured reproducibly by the cell culture assay used. These results are suitable as the starting point for the development of the neutralisation test using cell cultures.

Formation of cytotoxins by enteric Campylobacter in humans and animals.

Campylobacter (C.) jejuni from persons suffering from diarrhoea, from organs of poultry, C. jejuni and C. fetus ssp. fetus from the gastrointestinal tract of calves and adult cattle as well as a number of reference strains were examined for cytotoxin formation in a CHO-K1 cell culture test. During evaluation, three morphologically different pictures were observed. The first cytotoxin caused a formation of strikingly large, rounded or polymorphic and elongated cells which was associated with reduced growth. The progressive morphological changes corresponded to those described for the Cytolethal Distending Toxin (CLDT) and were assigned to it. The second cytotoxin produced a rounding of cells without a change in their size while at the same time, growth was reduced. In analogy to CLDT, this toxin was termed Cytolethal Rounding Toxin (CLRT). A third morphological picture consisted of cell changes characterized by enlarged polymorphic as well as by small rounded cells. These cell changes were considered as being distinct from the above mentioned ones and referred to as CLTD/CLRT effect. In none of the 39 Campylobacter strains isolated from humans and calves with diarrhoea, a noteworthy cytotonic activity could be detected that would indicate the presence of an enterotoxin.

[Detection of Clostridium perfringens toxins on cell cultures]. / Nachweis von Clostridium-perfringens-Toxinen auf Zellkulturen.

An important prerequisite for the possible application of cell culture systems as an in vitro method for the potency test of C. perfringens vaccines is the detection of cytotoxic effects of non-neutralized toxins on cell cultures. For this purpose eight cell lines were investigated in terms of the sensitivity to toxins using microscopy and the MTT assay. The results suggest that the MDCK cell line is a sensitive and specific detection system for epsilon-toxin. The VERO cell line is a suitable indicator for the detection of beta-toxin. Another task was the selection of a sensitive method for the analysis of cytotoxic effects of the toxins used. The neutral red and the tetrazolium-(MTT)-assays were compared for three cell lines (MDCK, FBTR, MA 104). The MTT assay was proved to be the optimal test system under our conditions.

Fermenter cultivation of intestinal spirochaetes.

We cultivated 6 strains of intestinal spirochaetes in a laboratory fermenter under constant conditions (medium, physical-chemical parameters) in a permanent gas flow. It has been possible to demonstrate that the application of the fermenter technique for the cultivation of spirochaetes is a suitable method which can be easily standardized.