Biomedical subjects

E Liebana

Publications and source records attributed to E Liebana.

26 records · Page 2Linked to original sources

PubMed2002

Use of molecular fingerprinting to assist the understanding of the epidemiology of Salmonella contamination within broiler production.

1. We analysed Salmonella isolates by conventional sero- and phage-typing, as well as by molecular techniques within the broiler production chain in two integrated companies. The most prevalent serovars were selected for genetic fingerprinting. 2. Isolates were first screened by plasmid profiling; subsequently, the most common plasmid types within the prevalent zoonotic serovars (enteritidis and typhimurium) and S. agama were further characterised by PstI-SphI ribotyping, and XbaI pulsed field gel electrophoresis (PFGE). 3. Salmonella binza, S. kedougou, and S. 4,12:d:- were endemic in the feed mills over long periods of time, and a variety of plasmid types for each of the serovars were found in the premises. 4. A similar situation was found with S. binza and S. senftenberg within the hatchery in company B. The Salmonella serovars which were resident in those locations were also the ones most widely distributed throughout the broiler flocks. 5. Plasmid profiling was useful to subdivide clusters of isolates within serovars, but for each serovar a high percentage (36 to 79%) of the isolates tested fall within a prevalent plasmid type. 6. A more detailed genetic analysis of the isolates by a multiple typing approach allowed for further strain differentiation, and allowed some epidemiological conclusions to be drawn.

Animals↗

PubMed2002

Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease.

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease XbaI, while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously. The 207 E. coli O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E. coli O157 isolates in the study (Simpson's index of diversity [D] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E. coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E. coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.

Animals↗

PubMed2001

Lipopolysaccharide O-chain microheterogeneity of Salmonella serotypes Enteritidis and Typhimurium.

Variability in the lipopolysaccharide (LPS) of the two most prevalent Salmonella serotypes causing food-borne salmonellosis was assessed using gas chromatography analysis of neutral sugars from 43 Salmonella enterica serovar Enteritidis (S. Enteritidis) and 20 Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates. Four substantially different types of O-chain chemotypes were detected using cluster analysis of sugar compositions; these were low-molecular-mass (LMM) LPS, glucosylated LMM LPS, high-molecular-mass (HMM) LPS and glucosylated HMM LPS. Nineteen out of 20 S. Typhimurium isolates yielded glucosylated LMM. In contrast, S. Enteritidis produced a more diverse structure, which varied according to the source and history of the isolate: 45.5% of egg isolates yielded glucosylated HMM LPS; 100% of stored strains lacked glucosylation but retained chain length in some cases; and 83.3% of fresh isolates from the naturally infected house mouse Mus musculus produced glucosylated LMM LPS. A chain length determinant (wzz) mutant of S. Enteritidis produced a structure similar to that of S. Typhimurium and was used to define what constituted significant differences in structure using cluster analysis. Fine mapping of the S. Enteritidis chromosome by means of a two-restriction enzyme-ribotyping technique suggested that mouse isolates producing glucosylated LMM LPS were closely related to orally invasive strains obtained from eggs, and that stored strains were accumulating genetic changes that correlated with suppression of LPS O-chain glucosylation. These results suggest that the determination of LPS chemotype is a useful tool for epidemiological monitoring of S. Enteritidis, which displays an unusual degree of diversity in its LPS O-chain.

Animals↗

PubMed2001

Diversity of strains of Salmonella enterica serotype enteritidis from English poultry farms assessed by multiple genetic fingerprinting.

Reliable and sufficiently discriminative methods are needed for differentiating individual strains of Salmonella enterica serotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism was PstI-SphI ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of XbaI-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins of Salmonella serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.

Animals↗

PubMed2001

Molecular typing of Salmonella serotypes prevalent in animals in England: assessment of methodology.

Salmonella enterica serotypes Derby, Mbandaka, Montevideo, Livingstone, and Senftenberg were among the 10 most prevalent serotypes isolated from farm animals in England and Wales in 1999. These serotypes are of potential zoonotic relevance; however, there is currently no "gold standard" fingerprinting method for them. A collection of isolates representing the former serotypes and serotype Gold Coast were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), and ribotyping. The success of the molecular methods in identifying DNA polymorphisms was different for each serotype. Plasmid profiling was particularly useful for serotype Derby isolates, and it also provided a good level of discrimination for serotype Senftenberg. For most serotypes, we observed a number of nontypeable plasmid-free strains, which represents a limitation of this technique. Fingerprinting of genomic DNA by ribotyping and PFGE produced a significant variation in results, depending on the serotype of the strain. Both PstI/SphI ribotyping and XbaI-PFGE provided a similar degree of strain differentiation for serotype Derby and serotype Senftenberg, only marginally lower than that achieved by plasmid profiling. Ribotyping was less sensitive than PFGE when applied to serotype Mbandaka or serotype Montevideo. Serotype Gold Coast isolates were found to be nontypeable by XbaI-PFGE, and a significant proportion of them were found to be plasmid free. A similar situation applies to a number of serotype Livingstone isolates which were nontypeable by plasmid profiling and/or PFGE. In summary, the serotype of the isolates has a considerable influence in deciding the best typing strategy; a single method cannot be relied upon for discriminating between strains, and a combination of typing methods allows further discrimination.

Animals↗

PubMed2001

Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype Typhimurium DT104 isolates.

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC-->AAC) mutation, an Asp-87-to-Gly (GAC-->GGC) mutation, and a Ser-83-to-Phe (TCC-->TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC-->TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC-->TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

Animals↗

PubMed2000

In vitro T-cell activation of monocyte-derived macrophages by soluble messengers or cell-to-cell contact in bovine tuberculosis.

The macrophage plays a dual role in tuberculosis, promoting not only protection against mycobacteria, but also survival of the pathogen. Macrophages inhibit multiplication of mycobacteria but also act in concert with lymphocytes through presentation of antigens to T cells. Studies in animal and human infections have suggested a correlation of in vitro growth rates of mycobacteria with in vivo virulence, using uracil uptake to assess mycobacterial metabolism. This study found that blood-derived, non-activated bovine macrophages were capable of controlling Mycobacterium bovis bacillus Calmette-Gurin growth for up to 96 hr, but were permissive to intracellular growth of virulent M. bovis. The present investigation compared the in vitro modulation of these macrophage activities by cytokine-rich T-cell supernatants or cell-to-cell contact. On the one hand, treatment of cultured monocytes with mitogen-produced T-cell supernatants promoted morphological changes suggestive of an activation status, enhanced the antigen presentation capabilities of monocytes and up-regulated major histocompatibility complex class II expression. However, this activation was not associated with enhanced anti-M. bovis activity. On the other hand, incubation of infected monocytes with T-cell populations resulted in proportionally increased inhibition of M. bovis uracil uptake. This inhibition was also seen using cells from uninfected animals and indicated the necessity for cell-to-cell contact to promote antimycobacterial capability.

Animals↗

PubMed2000

Cellular interactions in bovine tuberculosis: release of active mycobacteria from infected macrophages by antigen-stimulated T cells.

The outcome of Mycobacterium bovis infections depends on the interactions of infected macrophages with T lymphocytes. Several studies in humans and in mouse models have suggested an important role for cytotoxicity in the protective immune response to mycobacterial infections, and both CD4+ and CD8+ T cells have been shown to elicit appropriate cytolytic activity. The present study investigated in vitro interactions of T cells with M. bovis-infected macrophages in bovine tuberculosis. The results showed that following interaction with antigen-stimulated peripheral blood mononuclear cells (PBMC) from infected cattle, there was an increased presence of M. bovis in the extracellular compartment of infected macrophage cultures, as measured by incorporation of [3H]uracil into mycobacterial RNA. Furthermore, out of a panel of T-cell clones from infected cattle, it was found that a higher proportion of CD8+ clones produced an increase in the number of metabolically active extracellular M. bovis organisms compared with CD4+ clones. Finally, a positive correlation between percentage of antigen-dependent release of mycobacteria and total uracil uptake by M. bovis within culture systems was detected. This could be regarded as an indication of preferential intracellular control of mycobacteria by activated macrophages.

Animals↗