Two hundred nineteen Clostridium difficile isolates from 22 serogroups were screened for changes in the genes coding for toxin B (tcdB) and toxin A (tcdA). Parts of the toxin genes were amplified, and the PCR fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the tcdB gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less heterogeneous in the tcdA gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in tcdA, and four new types of shortenedtcdA genes are described. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly found are A1, A15, E, and X. Testing of variability inC. difficile toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.

S u rn m a r yThe epidemiology of campylobacteriosis was studied in broilers. Although two flocks of parent hens were infected, their progeny in 10 broiler flocks was not found infected before the 21st day of age. The proportion of infected flocks in winter and in spring was much smaller than in summer. Uninfected flocks had a higher average weight and production index but also higher feed conversion and losses. Domestic and free-living animals, found on the farm premises, and contaminated farmer's boots were sources from which the broilers could have been infected. C. jejunilcoli was not isolated from the feces of healthy farmers and their family members, litter, water and feed. C. jejuni biotype I according to Lior's biotyping scheme was found to be the most common. Using antisera raised against 25 Penner reference strains, serogroups 1, 2, 4, 5, 7, 9, 11, and 24 were found both in chicken and in possible sources of their infection.

We have used six independent polymerase chain reactions (A1-A3 and B1-B3) for amplification of the entire sequence of the two toxin genes tcdA and tcdB of several Clostridium difficile strains. With this approach we have detected (1) restriction site polymorphisms which are distributed all over the genes, and (2) deletions that could be found only in tcdA. Characteristic differences between strains were mainly focused to the 5' third of tcdB (B1 fragment) and/or the 3' third of tcdA (A3 fragment). The possible use of our approach for typing of C. difficile toxin genes is discussed.

Terrestrial isopods Porcellio scaber (Crustacea) were fed for five weeks on food contaminated by 250, 500, or 1,000 microg of Cd/g or for 10 d on diets with 50 or 250 microg Cd/g food. In both experiments, fecal production rate and colony forming units (CFUs) in the guts were determined. In addition, at the end of 10 d, each distinct colony morphotype obtained in gut samples was purified and characterized. Isolates were separated into 25 groups based on morphological and biochemical characteristics. These bacterial groups were used as units for calculating Shannon equitability indices (J) for each gut. The relative frequencies of the 25 bacterial units were determined in both cadmium groups (50 or 250 microg Cd/g food) and in the control. Cadmium-induced perturbations observed in the gut microbial communities were (1) increased number of morphologically distinct bacterial isolates in the group fed low-cadmium-dosed food (50 microg Cd/g) and reduced number of morphologically distinct bacterial isolates in the group fed high-cadmium-dosed food (250 microg Cd/g) compared with the control, (2) increased or decreased relative frequencies of almost all 25 bacterial units provoked by cadmium-contaminated food, (3) time-dependent increased numbers of gut CFUs in cadmium-fed animals (dose dependence was not observed), and (4) significant changes in community structure described by Shannon equitability indices at lower levels of food contamination (50 microg Cd/g) only. Gut microflora parameters are proposed as additional endpoints in the standardized single-species toxicity test with the terrestrial isopod P. scaber as a means of increasing the ecological relevance of the results.

Isolates of Campylobacter jejuni and Campylobacter coli isolated from two flocks of parent hens and their progeny which were followed from hatch to slaughter in 10 different farms within a 6-month period in the area of Ljubljana, Yugoslavia were bio- and serotyped. They were compared to those isolated from diarrheic patients within the same period of time. C. jejuni biotype I of Lior's biotyping scheme was found most predominant. Using 25 unabsorbed antisera raised against live C. jejunicoli cultures, 62.2% and 44.8% of the isolates from patients and chickens, respectively, could be serotyped. Penner sergroups (PG) 1, 2, 5, 7, 9, and 22 were found common to both patients and chickens. PG 2 was the most common isolate. PG 8, which was the second most frequently isolated serogroup from patients was not isolated from chickens. No Campylobacters were isolated from 71 farm family members