Molecular epidemiological tools for Salmonella Dublin typing

Abstract A total of 32 strains of Salmonella Dublin recovered from cattle were differentiated by electrophoretic typing of their esterases (zymotyping), restriction fragment length polymorphism of ribosomal DNA (ribotyping), arbitrarily primed PCR (AP-PCR) using five primers, PCR based on repetitive extragenic palindromic sequences (REP-PCR) and PCR based on enterobacterial repetitive intergenic consensus sequences (ERIC-PCR). ERIC-PCR and REP-PCR each gave one type, zymotyping gave three, AP-PCR gave five and ribotyping gave seven types. Combination of ribotyping and AP-PCR produced a total of 11 types, whereas 14 different types were obtained by all five methods. Thus a combination of several methods enhanced the discrimination of cattle-adapted strains among the genotypically homogeneous serovar Salmonella Dublin.     

Pulsed-field electrophoretic fingerprinting of Salmonella indiana and its epidemiological applicability

Eight Xba I-generated pulsed-field profile (PFP) types and four subtypes within one of the most common PFP types have been identified in Salmonella indiana from patients, poultry and human food in England and Wales in the three-year period from January 1994 to December 1996. Two PFP types have predominated, PFP X1 and PFP X2. Although the PFP X1 type was identified throughout the study period, the PFP X2 type was not identified until late 1995, subsequently becoming the most common PFP type in humans in the first six months of 1996 with a significant distribution in elderly patients. It is concluded that PFGE can be used in support of epidemiological investigations for the subdivision of Salm. indiana . Furthermore, as both conditions and interpretation criteria can be easily standardized, it is suggested that for many salmonella serotypes, PFGE can provide the basis for a definitive scheme of genotypic subtyping suitable for epidemiological investigations at both a national and international level.     

Pulsed-field electrophoretic fingerprinting of Salmonella indiana and its epidemiological applicability

Eight Xba I-generated pulsed-field profile (PFP) types and four subtypes within one of the most common PFP types have been identified in Salmonella indiana from patients, poultry and human food in England and Wales in the three-year period from January 1994 to December 1996. Two PFP types have predominated, PFP X1 and PFP X2. Although the PFP X1 type was identified throughout the study period, the PFP X2 type was not identified until late 1995, subsequently becoming the most common PFP type in humans in the first six months of 1996 with a significant distribution in elderly patients. It is concluded that PFGE can be used in support of epidemiological investigations for the subdivision of Salm. indiana. Furthermore, as both conditions and interpretation criteria can be easily standardized, it is suggested that for many salmonella serotypes, PFGE can provide the basis for a definitive scheme of genotypic subtyping suitable for epidemiological investigations at both a national and international level.     

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.     

Various epidemiological aspects of the study of plasmids in Salmonella drug-resistance

Distribution of drug resistance in Salmonella isolated from different sources was studied over time. The strains of S. typhimurium characterized by more pronounced resistance to antibiotics were mainly isolated from infants. Circulation of such strains in animals was extremely rare. Some genetic markers of such pathogens were investigated.     

Molecular fingerprinting of Salmonella serotype Glostrup

Thirteen isolates of Salmonella serotype Glostrup (antigenic formula, 6.8:z10:e,n,z15) from various sources and countries were analysed by ribotyping and IS200 fingerprinting. Both methods provided a high index of strain discrimination by allowing detection of three ribotypes and eight IS200 fingerprints which, though generally related, were readily distinguishable. The findings of this analysis confirm the usefulness of ribotyping and IS200 fingerprinting for studying the epidemiology of rarely isolated salmonellae of serogroup C.     

Optimization of RAPD for fingerprinting Salmonella

Random amplification of polymorphic DNA (RAPD) is proving to be a useful technique in studying the epidemiology of micro-organisms. The technique can be troublesome and time consuming to establish due to the essentially empirical approach to optimization. By standardization of certain parameters and use of a commercially available PCR buffer optimization kit, a particularly promising primer was identified and RAPD conditions for a highly discriminatory and reproducible characterization of Salmonella isolates was achieved. In addition, a technique to obtain reproducible RAPD fingerprints of Salmonella isolates without the need to purify genomic DNA is described.     

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.     

Bacteriophage typing of Salmonella weltevreden

Salmonella weltevreden has been found to be one of the commonest Salmonella serotypes isolated from diverse sources in India and has also been isolated in a number of other countries. A phage typing scheme was developed for this serotype using a set of six typing phages. These phages had been selected out of 146 phage strains isolated and purified from stool samples of man, laboratory animals and other animals, sewage and surface water sources, and the lytic mutants of temperate phages from S. weltevreden.The phage typing scheme was applied systematically to type the 946 strains from India isolated during 1958–1974 and 148 strains originating from Australia, Burma, England, Gan Island, Holland, Hong Kong, Malaysia, New Zealand, Papua New Guinea, The Philippines, Thailand, The United States and Vietnam during 1953–1971. The scheme was particularly studied to evaluate its utility in mapping the epidemiologically related strains from various sources.The S. weltevreden strains could be classified into ten phage types. Phage types 2 and 7 were found exclusively amongst Indian strains, type 6 from Vietnam and type 8 from Burma, Thailand and Vietnam. Phage types were found to be stable and consistent with the independent epidemiological data available.     

Forensic applications of DNA fingerprinting

In many ways, DNA profiling technology is very similar to the conventional techniques used for forensic identification. As with, for example, blood grouping techniques, the molecular characteristics of the scene of crime sample may be determined and compared with those of the scene of reference samples from suspects and victim. If the molecular characteristics of the crime sample and the suspect are different, then they cannot be from the same person, whereas if they match, then the possibility remains that they may be from a single source. Similar material, such as blood or semen stains, may be used for both biochemical and genetic tests, and the main applications of identification and relationship testing are shared by both techniques. At this point, the similarity ends; DNA profiling has the following characteristics:

1. It is more sensitive, being able to generate sound data from only a tiny amount of even partially degraded biological material.
2. It is capable of resolving mixtures of semen or tissue from up to several individuals.
3. It has a far greater power of discrimination between individuals—sometimes up to 1 millionfold higher than conventional techniques.
4. It provides considerably more information on the nature of relationships, particularly in cases of incest.

As such, the technique represents a quantum leap in forensic identification and relationship testing.     

Viral infections and cancer: epidemiological aspects.

Viral infections represent one of the areas in which cancer research has made the greatest advances in the last 20 years. In 1981, only two viruses were known to cause human cancer, i.e., the Epstein-Barr virus (EBV) and the hepatitis B virus (HBV). By 1995, it was estimated that approximately 15% of all cancers occurring world-wide were attributable to viral infections, and the oncogenic role of seven viruses [i.e., EBV, HBV, hepatitis C virus (HCV), human papillomavirus (HPV), human immunodeficiency virus (HIV), human herpesvirus-8 (HHV-8), and human T cell leukemia virus type I (HTLV-I)] has been well-established. In this paper, the epidemiological evidence concerning some of the major aspects of the association between viruses and cancer are summarised.