Milk concentration improves Bluetongue antibody detection by use of an indirect ELISA.

A national Bluetongue antibody surveillance in cattle through bulk milk was conducted in Switzerland between July 2007 and June 2008. Using ID Screen Bluetongue Milk ELISA (ID VET, Montpellier, France), samples from 15 out of 210 dairy farms at least once gave a positive result. In only three of these herds bluetongue positive animals were found. Therefore, specificity for bulk milk was not as good as expected and when individual milk samples were tested, it was even lower. As further investigations of positive results were time-consuming and no other ELISA was available at that time, we aimed at discriminating false from true positive samples with a confirmatory test using a protein precipitation method followed by retesting with the same ELISA. Additionally, we examined whether testing of single milk samples can reliably be used to assess status of cows, and whether sampling at the beginning or at the end of milking, as well as freezing and thawing of the milk could influence the performance of the test. Screening with ID VET milk ELISA and confirmatory testing after protein precipitation yielded a clear increase of specificity without any loss of sensitivity in both bulk and single milk samples. This testing scheme allowed minimizing follow-up investigations by blood testing. Antibody levels in plasma and milk showed a good correlation. Tested by logistic regression, none of the possible influencing factors (time point of sample collection, freezing, or milk content of the samples) had a significant influence on the test performance.

Activatable Shiga toxin 2d (Stx2d) in STEC strains isolated from cattle and sheep at slaughter.

Shiga toxin producing Escherichia coli (STEC) harbouring the stx(2d-activatable) gene and expressing the mucus- and elastase-activatable phenotype have been associated with severe outcomes of human disease. However, there is limited data available on the occurrence of such strains in livestock reservoirs. In this study, we analyzed 11 STEC strains isolated from healthy cattle and sheep at slaughter that were originally detected to contain the stx(2c) allele, for the presence of the stx(2d-activatable) genotype. Ten of the eleven strains displayed the stx(2d-activatable) genotype as determine by PstI restriction fragment length polymorphism (RFLP) of 890-bp fragments of their stx genes. However, only in 6 of the 10 strains whose stx genes were sequenced, the presence of stx(2d-activatable) could be confirmed based on the predicted amino acid sequence of their StxA subunits; the remaining four strains contained Stx2c A subunit. Five of the six strains which contained stx(2d-activatable) displayed the activatable phenotype on Vero cells. Genes for adhesins such as the outer membrane protein intimin (eae), which is essential for the intimate attachment and the formation of attaching-and-effacing lesions on intestinal epithelial cells, or the STEC autoagglutinating adhesin (saa), potentially important in eae-negative STEC, were not detected. Moreover, all the strains tested negative for EHEC-hlyA encoding enterohaemorrhagic E. coli (EHEC) hemolysin. To our knowledge, this is the first study that reports the presence of STEC harbouring stx(2d-activatable) and producing the activatable Stx2d in fecal samples of sheep. Therefore both cattle and sheep are reservoirs of such strains and potential sources of human infections. This is of particular importance, because in contrast to other eae-negative STEC, strains producing Stx2d(activatable) may cause severe diseases such as bloody diarrhoea and haemolytic uremic syndrome in humans.

Comparison of two chromogenic media and evaluation of two molecular based identification systems for Enterobacter sakazakii detection.

BACKGROUND: Enterobacter sakazakii is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. The current FDA detection method includes two enrichment steps, the subculturing of the second enrichment broth on a selective agar (VRBG), a further subculturing of selected grown colonies on TSA and the subsequent biochemical identification of yellow-pigmented colonies by API20E. However, there is a strong need for simplified methods for isolation and identification of E. sakazakii. In this study, two chromogenic media, which allow to indicate presumptive E. sakazakii colonies by the alpha glucosidase activity, as well as a newly developed 1,6-alpha-glucosidase based conventional PCR assay and a rRNA oligonucleotide probe based commercial test system for identification of presumptive E. sakazakii were evaluated on 98 target and non-target strains. The methods were compared with respect to specificity aspects. RESULTS: A total of 75 presumptive E. sakazakii and 23 non-target strains were analysed by using chromogenic media, alpha-glucosidase based PCR assay, and the VIT assay. For most presumptive E. sakazakii strains on the chromogenic media, the PCR and VIT assay confirmed the identification. However, for a number of presumptive E. sakazakii isolates from fruit powder, the alpha-glucosidase PCR and VIT assay did not correspond to the typical E. sakazakii colonies on DFI and ESIA. Further characterization by API32E identification, phylogenetic analysis of partial 16S rRNA sequences and ribotyping strongly suggested, that these strains did not belong to the species E. sakazakii. The newly developed alpha-glucosidase based PCR assay as well as the commercially available VIT Enterobacter sakazakii identification test showed an excellent correlation with the 16S rRNA data, and are thus well suited for identification of E. sakazakii. CONCLUSION: The results indicate that presumptive colonies on ESIA and DFI media need further species identification. Both evaluated molecular methods, the alpha-glucosidase PCR and the 16S RNA in situ hybridisation test (VIT), although based on completely different target regions and methodologies performed equally well in terms of specificity.