SummaryMycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patientderived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the nonvirulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.

SUMMARYFour UK strains of three different serotypes were found to differ by only 2-3% of their S1 amino acids. The S1 sequences were also very similar to those of three Dutch isolates (D207, D274 and D3896), the greatest difference between two of the seven isolates being 4.4%. The few amino acid differences between the seven isolates were located largely between residues 19-122 and 251-347 of the mature S1 subunit.The seven isolates could be differentiated using 16 monoclonal antibodies in an enzymelinked immunosorbent assay. Some virus neutralizing (VN) antibody-inducing epitopes were common to all seven isolates even though the strains had been differentiated into three serotypes by polyclonal sera. The results indicate that the most antigenic of the VN antibody-inducing epitopes are formed by very few amino acids and that these occur in the first and third quarters of the S1 subunit. We suggest that serology-based epizootiological studies of IBV should, therefore, be augmented by the inclusion of nucleic acid sequencing and/or monoclonal antibody analysis.

Broilers and broiler meat products are highly contaminated with extended spectrum beta-lactamase (ESBL) or plasmid-mediated AmpC beta-lactamase producing Escherichia coli and are considered to be a source for human infections. Both horizontal and vertical transmission might play a role in the presence of these strains in broilers. As not much is known about the presence of these strains in the whole production pyramid, the epidemiology of ESBL/AmpC-producing E. coli in the Dutch broiler production pyramid was examined. Cloacal swabs of Grandparent stock (GPS) birds (one−/two-days (breed A and B), 18 and 31 weeks old (breed A)), one-day old Parent stock birds (breed A and B) and broiler chickens of increasing age (breed A) were selectively cultured to detect ESBL/AmpC-producing isolates. ESBL/AmpC-producing isolates were found at all levels in the broiler production pyramid in both broiler breeds examined. Prevalence was already relatively high at the top of the broiler production pyramid. At broiler farms ESBL/AmpC producing E. coli were still present in the environment of the poultry house after cleaning and disinfection. Feed samples taken in the poultry house also became contaminated with ESBL/AmpC producing E. coli after one or more production weeks. The prevalence of ESBL/AmpC-positive birds at broiler farms increased within the first week from 0–24% to 96–100% independent of the use of antibiotics and stayed 100% until slaughter. In GPS breed A, prevalence at 2 days, 18 weeks and 31 weeks stayed below 50% except when beta-lactam antibiotics were administered. In that case prevalence increased to 100%. Interventions minimizing ESBL/AmpC contamination in broilers should focus on preventing horizontal and vertical spread, especially in relation to broiler production farms.

Neutralizing monoclonal antibodies directed against five antigenic sites on the spike (S) S 1 glycopolypeptide of avian infectious bronchitis virus (IBV) were used to select neutralization-resistant variants of the virus. By comparing the nucleotide sequence of such variants with the sequence of the IBV parent strain, we located five antigenic sites on the amino acid sequence of the S 1 glycopolypeptide. The variants had mutations within three regions corresponding to amino acid residues 24 to 61, 132 to 149 and 291 to 398 of the S1 glycopolypeptide. The location of three overlapping antigenic sites on the IBV spike protein was similar to the location of antigenic sites on the spike protein of other coronaviruses.

Monoclonal antibodies (MAbs) directed against structural proteins of infectious bronchitis virus (IBV) were produced to analyse the antigenic structure of this virus. Competitive binding of enzyme-labelled and unlabelled MAbs to IBV peplomer protein was analysed in an antibody binding assay to test the relatedness of the epitopes defined by the MAbs. Based on the competition groups, eight epitope clusters were defined (S-A to S-H); six of these clusters (S1-A to S I-F) were located on the S1 subunit and two (S2-G and S2-H) on the $2 subunit of the peplomer protein.Epitope clusters S 1-A and S1-B overlapped extensively. The biological activities of the MAbs were determined and correlated to the epitope clusters. Monoclonal antibodies directed against epitope clusters S 1-A to S1-E and one MAb directed against cluster S2-G moderately to strongly neutralized IBV at titres higher than 2 log~o, whereas the remaining MAbs, directed against S1 and $2, neutralized at titres lower than 2 log~o. One MAb, directed against cluster S1-D, inhibited the agglutination of chicken erythrocytes.

This is the first study among a large collection of Dutch companion animals and horses characterizing ESBL/AmpC-producing isolates. A similarity in resistance genes and E. coli STs among these isolates and isolates from Dutch poultry and humans may suggest exchange of resistance between different reservoirs.

Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.

-We have amplified, cloned and sequenced (part of) the open reading frame of the S1 segment encoding the σ C protein of avian reoviruses isolated from chickens with different disease conditions in Germany and The Netherlands during 1980 up to 2000. These avian reoviruses were analysed phylogenetically and compared with sequences of avian reoviruses in the Genbank database. The avian reoviruses could be grouped in 5 different genotyping clusters and this classification was identical when the sequences were compared of the 5' end, the 3' end or the whole open reading frame of the σ C protein. Therefore sequencing of either part of the gene encoding the σ C protein seems to be reliable for classification. We were unable to identify a correlation between σ C sequences of the avian reoviruses and the disease condition they were isolated from. The sequences found in The Netherlands and in Germany are, like those in Taiwan, more dispersed than the known avian reovirus σ C sequences in the USA and Australia. We did not establish temporal or geographic differences in the avian reoviruses studied. avian reovirus / malabsorption syndrome / tenosynovitis / σ C gene / genotype