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ABSTRACTA desirable test to diagnose infections with Mycobacterium aviumsubsp. paratuberculosisfacilitates identification of infected cattle prior to the state of M. aviumsubsp. paratuberculosisshedding. This study aimed at adjusting a flow cytometry (FC)-based assay, using intact M. aviumsubsp. paratuberculosisbacteria as the antigen, for diagnosis of M. aviumsubsp. paratuberculosisinfections in calves. Serum samples were collected from experimentally infected (n= 12) and naturally exposed (n= 32) calves. Samples from five calves from positive dams were analyzed to determine the dynamics of maternal antibodies. Samples from adult cattle with defined infection status served as the standard (18 M. aviumsubsp. paratuberculosisshedders, 22 M. aviumsubsp. paratuberculosisfree). After preadsorption with Mycobacterium phlei, sera were incubated with M. aviumsubsp. paratuberculosisand M. aviumsubsp. aviumbacterial suspensions, respectively, followed by the separate detection of bovine IgG, IgG1, IgG2, and IgM attached to the bacterial surface. M. aviumsubsp. paratuberculosis-specific sample/positive (S/P) ratios were compared to enzyme-linked immunosorbent assay (ELISA) S/P ratios. In adult cattle, the FC assay for IgG1 had a sensitivity of 78% at a specificity of 100%. Maternally acquired antibodies could be detected in calves up to 121 days of life. While all but two sera taken at day 100 ± 10 postnatum from naturally exposed calves tested negative, elevated S/P ratios (IgG and IgG1) became detectable from 44 and 46 weeks postinoculation onwards in two calves infected experimentally. Even with the optimized FC assay, M. aviumsubsp. paratuberculosis-specific antibodies can only occasionally be detected in infected calves less than 12 months of age. The failure to detect such antibodies apparently reflects the distinct immunobiology of M. aviumsubsp. paratuberculosisinfections rather than methodological constraints.

Twenty verocytotoxigenic Escherichia coli (VTEC) O118 strains isolated between 1996 and 1998 from human patients in Germany were analysed for their serotypes, their virulence markers and their epidemiological relatedness. Three strains were typed as O118∶H12, these carried only the VT2d-Ount variant gene and were not associated with diarrhoea or haemolytic uraemic syndrome (HUS). Seventeen strains were serotyped as O118∶H16 or O118∶non-motile (NM). These carried all the genes for VT1, eae and EHEC-haemolysin. The O118∶H16/NM strains were from diarrhoea (13 cases) and HUS (2 cases). Sixteen of the patients were young infants and most infections were associated with a rural environment. Evidence for zoonotic transmission from cattle to humans was found in two cases. The epidemiological relationship between the human and bovine O118∶H16/NM isolates was indicated by homogeneous plasmid patterns and by very similar XbaI restriction patterns obtained by pulsed-field gel electrophoresis. VTEC O118∶H16/NM are emerging pathogens in Germany and should be classified as new enterohaemorrhagic E. coli (EHEC) types.

Verotoxin-producing Escherichia coli isolates from feces of healthy cattle were identified by DNA hybridization with verotoxin 1- and verotoxin 2-specific gene probes. Among 259 animals investigated, 28 (10.8%) were found to carry verotoxin-producing E. coli strains. Characterization of the verotoxin-producing isolates revealed a heterogeneous population in terms of serotype and toxin type. Nearly 40% of the strains belonged to serogroups known to be pathogenic for humans, i.e., O22, O39, O82, O91, O113, O116, O126, and O136. Two isolates from different bulls were identified as serotype O157:H7. Results obtained in this study indicate that cattle may be an important source of verotoxigenic E. coli involved in human disease.