Quantifying the sources of Salmonella on dressed carcasses of pigs based on serovar distribution.

Salmonella serotyping data, qualitatively described by van Hoek et al. (2012), were used to quantify potential sources of Salmonella in a Dutch pig slaughterhouse. Statistical tests to compare per-day Salmonella prevalence and serotyping data from multiple points in the chain were used to find transmission pathways. A statistical model based on serotyping data was developed to attribute Salmonella on dressed carcasses to the most likely source. Approximately two-third of dressed carcasses carrying Salmonella on the medial surface had been contaminated by house flora. For carcasses carrying Salmonella on the distal surface, transient Salmonella from incoming pigs was a more important source. The relevance of the different sources of Salmonella varied within and between sampling days. Results were compared to those of another modeling approach, in which Salmonella concentration data from the same samples were used (Smid et al., 2012). They mostly agreed. The approach chosen by an individual slaughterhouse depends on the data that are collected.

Development of a novel, simple and rapid molecular identification system for clinical Candida species.

Identification of clinical yeast isolates causing candidiasis is routinely performed by commercial yeast identification systems based on biochemical, morphological and physiological tests. These systems require 3-5 days and the proportion of identifications that are incorrect is high. Our novel and rapid molecular identification system for clinical Candida species is based on the analysis of restriction patterns obtained from PCR-generated ribosomal DNA sequences using five restriction enzymes. A software package (CandID) was designed to include a database of restriction fragment length polymorphism (RFLP) patterns for 29 Candida species. For 'in-house' validation, 122 clinical isolates that had previously identified in clinical laboratories were typed by this system. These clinical isolates were also independently re-identified by the API 20C AUX system. The ribosomal DNA RFLP database in the context of supporting analytical software allowed simple and rapid (1 work day) identification.

Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains.

For the detection of pathogenic Yersinia enterocolitica strains, a duplex PCR has been developed based on differences observed between the fingerprint profiles of pathogenic and non-pathogenic strains. The profiles were obtained by using a primer derived from the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences. From the sequence of one pathogen-specific amplified fragment, a discriminative primer has been designed bridging the sequence of the highly conserved core region and 3' end of the ERIC element. In combination with three other primers, all located within the detected open reading frame that resembled the sequence of the bipA gene, this primer was applied in a duplex PCR assay to simultaneously detect Y. enterocolitica and to discriminate between pathogenic and non-pathogenic strains. The same primer combinations were used in an on line rapid cycling real-time PCR assay. The used SYBR Green I format allowed for the easy translation of the PCR conditions and confirmation of the resulting amplicons. The time of analysis was reduced to approximately 60 min.

Molecular tools for the characterisation of antibiotic-resistant bacteria.

This review will discuss a number of molecular tools which are currently used as well as some innovative approaches for the characterisation of antibiotic-resistant bacterial strains. Various methods involved in the detection and characterisation of genes and mutations associated with antibiotic resistance and that are used for strain typing as part of epidemiological studies, are described. Furthermore, a few examples are discussed in which the results of both gene and strain characterisation are combined to investigate the underlying mechanism of the spread of antibiotic resistance. Some of the available molecular techniques are heavily supported by the existence of databases on the Internet. These databases either contain a fast growing amount of sequence information or a large number of allelic or fingerprint profiles. The current progress in applied DNA technology and the ongoing projects on the elucidation of the whole genomic sequence of bacterial species have lead and will further lead to the development and application of sophisticated new strategies for the analysis of antibiotic resistant bacterial strains.

Genomic typing of Listeria monocytogenes strains by automated laser fluorescence analysis of amplified fragment length polymorphism fingerprint patterns.

The genetic relationship between isolates of Listeria monocytogenes belonging to different serotypes was determined and the suitability of automated laser fluorescent analysis (ALFA) of amplified fragment length polymorphism (AFLP) fingerprints was assessed by genomic typing of 106 L. monocytogenes isolates belonging to serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 1, and 7. Digitised AFLP fingerprints were obtained that showed approximately 50 clearly distinguishable selectively amplified EcoRI/MseI bands for each strain. The coefficient of similarity between the profiles was determined by simple matching (Ssm). Based on these coefficients of similarity the investigated strains clustered in two genomic groups. The first group consisted of strains belonging to serotype 1/2a, 1/2c, 3a and 4a, while the second group was comprised of strains belonging to serotypes 1/2b, 3b, 4ab, 4b, 4e and 1. The average simple matching coefficient of similarity between strains of the second group was 92%, which was 4% higher than within group 1. Hence, the serotypes which are responsible for the majority of the listeriosis cases, 1/2a, 1/2b and 4b, fall into two distinct genetic groups, in concordance with their flagellar antigen type. The discriminatory power of AFLP in combination with automation of the analysis of the fingerprint profiles by ALFA makes AFLP-ALFA highly suitable for typing L. monocytogenes.

High-resolution genotyping of Salmonella strains by AFLP-fingerprinting.

High resolution AFLP fingerprinting, in which subsets of genomic restriction fragments are amplified by means of PCR, was used for the identification of different. Salmonella serotypes to investigate whether this technique is applicable in epidemiological studies. Seventy-eight different Salmonella strains comprising 62 serotypes were genetically identified by AFLP. Primer combination M00 (MseI primer without additional 3' nucleotides) and E11 (EcoRI primer with two additional 3' nucleotides) resulted in reproducible profiles containing approximately 50 bands. All serotypes were characterized by a unique profile. In addition, AFLP fingerprinting enabled phage type identification. Different strains previously identified as identical, using typing methods with lower resolution, could be distinguished, showing that AFLP fingerprinting is well suited for bacterial epidemiology and identification.

Occluded hemodialysis shunts: Dutch multicenter experience with the hydrolyser catheter.

PURPOSE: To evaluate mechanical thrombectomy of occluded hemodialysis access shunts with a recently developed hydrodynamic device. MATERIALS AND METHODS: Sixty-five thrombosed hemodialysis access shunts were treated in 49 patients. The shunts were of three types: Brescia-Cimino fistulas (24 procedures), polytetrafluoroethylene (PTFE; Gore-Tex) loop grafts (18 procedures), and manufactured homologous vein loop grafts (23 procedures). Clots were removed by means of aspiration caused by the hydrodynamic effect of a high-velocity flow of saline through the catheter (Venturi effect). RESULTS: Successful declotting was achieved in 58 of 65 (89%) attempts. Early reocclusion occurred in 11 shunts and was successfully treated by means of repeat thrombectomy in five instances. Procedure time averaged 1-1 1/2 hours. The primary patency was similar for the three types of access shunts (P = .09), with a median of 14 weeks (including the initial treatment failures). Assisted patency for polytetrafluoroethylene loop grafts was better than that for the two other types (P = .002). Complications were encountered in 10 of 65 (15%) cases. These included formation of a large local hematoma that resulted in loss of a Brescia-Cimino fistula, two instances of arterial embolization, and one case of pulmonary embolization of thrombus material. CONCLUSION: Effectiveness of mechanical thrombectomy of occluded hemodialysis access shunts with the described hydrodynamic device is similar to that of alternative treatments such as thrombolysis.

Association of endogenous avian viral and endogenous viral genes with feed conversion and six-week body weight in broilers.

The consistency of the effect of selection on the frequencies of endogenous avian viral (eav) and endogenous viral (ev) specific restriction fragment length polymorphism (RFLP) bands was studied in two broiler lines selected from a single base population and in an F2 population derived from a reciprocal cross of both lines. One broiler line (FC line) was selected for low feed conversion ratio and the other line (GL line) was selected for high 6-wk body weight. In the F2 population, the band frequencies were determined in groups representing separate tails of the distribution of two production traits, namely, low feed conversion ratio between 29 and 42 d of age and body weight at 42 d of age. The F2 population consisted of 288 females belonging to 24 full-sib families. To rule out family effects, the tails for these production traits were composed by either the best or by the worst female performer for each trait in each full-sib family. In total, 29 HindIII-eav, 34 MspI-eav, and 21 BamHI-ev bands could be distinguished by RFLP analysis. This report describes the influence of selection on 11 potentially interesting bands. Two bands, the 9.5-kb HindIII-eav and the 15-kb MspI-eav band, which were found both in higher frequencies in the parental FC line, were also found in higher (P < or = .05) frequencies in the F2 tail with a favorable feed conversion ratio. A third band, the 6.5-kb HindIII-eav band, present in lower frequencies in the parental GL line, was also present in lower (P < or = .05) frequencies in the F2 tail of birds with heavy body weight.

Discrepancy between Penner serotyping and polymerase chain reaction fingerprinting of Campylobacter isolated from poultry and other animal sources.

Thirty-four Campylobacter jejuni or coli strains, isolated from various livestock and darkling beetles from two Dutch poultry farms during different broiler production cycles, were subjected to Penner serotyping and polymerase chain reaction (PCR) fingerprint analysis. Ten different Penner serotypes were determined in the isolates. Visual scoring of the PCR fingerprints resulted in 14 clearly different profiles. Some strains with identical Penner serotypes exhibited different PCR fingerprints and conversely strains with different serotypes produced identical PCR fingerprints. Discrepancies between Penner serotyping and PCR fingerprinting were most obvious between isolates from different animal sources. Indications for the occurrence of genomic rearrangements were found. The inconsistency between serotyping and fingerprinting of Campylobacter strains suggests that conventional typing methods should be used in combination with fingerprinting if the epidemiological factors that contribute to Campylobacter colonization of live chickens are to be assessed reliably.

Polymerase chain reaction identification of Salmonella serotypes.

Seventy-seven Salmonella isolates comprising 61 different serotypes were subjected to polymerase chain reaction (PCR) fingerprinting using two primersets. Primerset L1/G1, amplifying the spacer regions between the 16S and 23S rRNA genes, resulted in simple PCR fingerprints. However, in some cases PCR amplification of different Salmonella serotypes with primerset L1/G1 resulted in identical fingerprint profiles. Fingerprints obtained with the ERIC primerset, that matches the enterobacterial repetitive intergenic consensus sequence, were more complicated but were serotype-specific. Consequently, fingerprinting with the ERIC primerset is applicable for typing Salmonella up to the serotype level. Fingerprinting with the L1 and G1 primers requires an additional treatment of the amplification product for accurate typing of salmonellas. Phage typing is not possible with either primerset.

The second human beta B2-crystallin gene is a pseudogene.

Comparison of the partial sequences of the human beta B2-1- and beta B2-2-crystallin genes with orthologous rat or calf sequences shows that the fourth exon sequence of the human beta B2-2 gene contains a one triplet deletion and a mutated splice acceptor site. No transcripts from the beta B2-2-crystallin gene could be detected in the human lens. These data suggest that the human beta B2-2-crystallin gene is a pseudogene.

Endogenous viral genes in thirteen highly inbred chicken lines and in lines selected for immune response traits.

Thirteen highly inbred lines of chickens of Leghorn, Spanish, and Egyptian Fayoumi origin, four partly inbred Leghorn lines selected for MHC alleles and immune response to GAT (Ir-GAT), and two replicated, noninbred Leghorn lines divergently selected for multiple immune response traits were subjected to molecular genotyping for endogenous viral (ev) gene sequences. In all highly inbred lines of Leghorn origin, ev1 alone or both ev1 and ev2 were observed. The Spanish and Fayoumi lines had three and five ev genes, respectively, most of which were not readily identifiable with standard Leghorn ev gene loci. The Leghorn lines selected for MHC and Ir-GAT had ev1 fixed in the population. Differences in ev3 and ev5 gene frequency were associated with Ir-GAT in the B1 haplotype, but not in the B19 haplotype. In the noninbred lines, which were divergently selected for multiple traits of immune responsiveness, ev6 and ev9 differed in frequency between lines, and both were in lower frequency in the lines selected for high immunoresponsiveness. These two ev genes are the only ones known in White Leghorns that have the gs-chf+ phenotype [expressing chicken helper factor (chf) but not expressing group-specific antigen (gs)].

Different endogenous viral loci in Cornish and White Plymouth rock chickens.

Endogenous viral (ev) loci were studied in three broiler lines. In 5 birds of each of line cw1 and line cw2 (White Plymouth Rock lines) 19 and 14, respectively, different SstI ev-junction fragments were found, while in 8 R line birds (Cornish type) 15 different Sst I junction fragments were found. Further characterization of the line R loci with a second restriction enzyme, BamHI, revealed that these junction fragments represent 25 different loci, of which at least 21 have not been reported previously. SstI RFLP analysis of progeny from crosses between chickens of the three broiler lines and White Leghorns demonstrated that within line R and cw1 approximately 90% of the ev loci were hemizygous. In line cw2 at least 50% of the ev loci were hemizygous. There was no evidence for polymorphic loci, and only two ev loci were found to be linked genetically. Intertype crosses revealed that overall differences in the RFLP patterns observed between Cornish, White Plymouth Rock and White Leghorn chicken lines were due to the presence of different ev loci in each of the lines rather than to polymorphism. The few shared ev loci always contained similar allelic fragments.

Variations in endogenous viral gene patterns in White Leghorn, medium heavy, White Plymouth Rock, and Cornish Chickens.

The endogenous viral (ev) gene patterns of White Leghorn (WL; 6 lines), medium heavy (MH; 4 lines), White Plymouth Rock (WPR; 8 lines), and Cornish types (2 lines) of commercial chickens were compared. Southern blot analysis of SstI-digested genomic DNA of 151 chickens revealed that the number of ev gene-containing fragments in a chicken from the MH, WPR, or Cornish type is about twice twice the number of that in WL chickens. Also, the number of hybridizing fragments of different size found within one line was twice as high in the broiler (on average 16.0 bands per line) and MH lines (20.5 bands per line) than in the WL lines (10.0 bands per line). In studies with subregion-specific probes, all ev fragments detected contained the env (3') part of the viral genome. Only eight ev fragments, found in 7 animals of 2 lines, lacked the gag (5') part of the viral genome. Studies with the ev-1-specific flanking probe, pGd111, revealed that ev-1 is commonly present in the DNA of the WL chickens, but not within the DNA of the WPR chickens. The results suggest that use of the standard nomenclature for the ev genes based on restriction fragment length is not feasible within the WPR, MH, and Cornish lines because of the complexity of the ev gene patterns found within these lines.

Human gamma-crystallin genes. A gene family on its way to extinction.

During hominoid evolution the gamma-crystallins of the lens have decreased in quantity as well as complexity, a change correlated with an increased water content of the lens. To trace the molecular basis for the decrease in gamma-crystallin gene expression, we have characterized the structure and expression of the human gamma-crystallin gene family. We show that the human gamma-crystallin gene family consists of six complete genes (gamma A, gamma B, gamma C, gamma D, psi gamma E and psi gamma F) and one second exon fragment, the gamma G gene. Model experiments showed that, although the gamma G sequence is bordered by consensus splice sites, it is most likely transcriptionally inactive in the lens. In the human embryonic lens the gamma C and gamma D genes accounted for 81% of the gamma-crystallin transcripts, the gamma A gene contributed 14% and the gamma B gene only 5%. The composition of the gamma-crystallin mRNA pool changed only after birth, with the gamma D transcript as the only detectable transcript at ten years of age. The relative activities of the gamma A, gamma C and gamma D promoters in a transient expression system were in agreement with the ratio of their in vivo RNA levels, suggesting that the difference in accumulation of these transcripts is due to differences in the rate of transcription. The gamma B promoter was much more active than expected and had lost its tissue-specificity. Model experiments showed that the low yield of the gamma B transcript is due to post-transcriptional processes, most likely RNa instability mediated by third exon sequences. Together with previous data, our results show that the decrease in expression of the gamma-crystallin genes in the human lens is the consequence of gene loss (gamma G), inactivation of coding sequences (psi gamma E and psi gamma F), decrease in rate of transcription (gamma A), increase in rate of RNA turn-over (gamma B) and a delay in the onset of transcription during development.

Crystallin gene expression during rat lens development.

The analysis of the developmental pattern of the alpha A-, alpha B-, beta B1-, beta B2-, beta B3-, beta A3/A1-, and beta s-crystallin genes during fetal and postnatal development of the rat shows that the differential regulation of crystallin synthesis relies on differential gene shutdown rather than differential gene activation; that is, all crystallin genes are active during early development but turn off at different stages. The only two exceptions to this rule are the alpha B- and beta s-crystallin genes. The alpha B-crystallin gene transcript becomes first detectable at 18 days of fetal development, while the beta s-crystallin gene appears to be active only in the postnatal period. We also determined the absolute numbers of the alpha A-, alpha B-, beta B1-, beta B2-, beta B3-, beta A3/A1-, beta s-, and gamma-crystallin gene transcripts present in the lens at various times after birth. Comparison of these RNA data with the published protein data shows that the alpha B- and beta B2-crystallin RNAs are relatively overrepresented, suggesting the possibility that these two RNA species are not used as efficiently as other crystallin mRNAs. Examination of the known (hamster) alpha B-crystallin sequence and elucidation of the (rat) beta B2-crystallin sequence yielded no evidence for aberrant codon usage. These two RNAs have one sequence motif in common: they are the only crystallin mRNAs in which the translation initiation codon is preceded by CCACC.

Different evolution rates within the lens-specific beta-crystallin gene family.

We have determined the sequence of a rat beta A3/A1-crystallin complementary DNA (cDNA) clone and the (partial) sequence of the human beta B3-crystallin gene. Calculation of the ratio of silent to nonsynonymous substitution between orthologous beta A3/A1-, beta B3-, and other beta- and gamma-crystallin sequences revealed that the region encoding the two globular domains of the beta A3/A1-crystallin sequence is the best conserved during evolution, much better than the corresponding region of the beta B1-, beta B3-, or the gamma-crystallin sequences, and even better (at least in the rodent/frog comparison) than the well-conserved alpha A-crystallin sequence. Remarkably, the rate of change of the beta A3/A1-crystallin coding sequence does not differ in the rodent and primate lineages, in contrast with previous findings concerning the evolution rates of the alpha A- or gamma-crystallin sequences in these two lineages. Comparison of the regions that encode the four motifs of the beta-crystallin between orthologous mammalian sequences showed that the extent of nonsynonymous substitution in each of these four homologous motif regions is the same. However, when the orthologous beta-crystallin genes of more distantly related species (mammals vs chicken or frog) are compared, the extent of non-synonymous substitution is higher in the regions encoding the external motifs I and III than in the regions encoding the internal motifs II and IV. This phenomenon is also observed when paralogous members of the beta/gamma-crystallin supergene family are compared.

The gamma-crystallin gene families: sequence and evolutionary patterns.

The gamma-crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the gamma-crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat gamma-crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.