F. A. Clifton-Hadley

Intestinal carriage of verocytotoxigenic Escherichia coli O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica, in cattle, sheep and pigs at slaughter in Great Britain during 2003.

An abattoir survey was undertaken to determine the prevalence of foodborne zoonotic organisms colonizing cattle, sheep and pigs at slaughter in Great Britain. The study ran for 12 months from January 2003, involved 93 abattoirs and collected 7703 intestinal samples. The design was similar to two previous abattoir surveys undertaken in 1999-2000 allowing comparisons. Samples were examined for VTEC O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica. The prevalence of VTEC O157 faecal carriage was 4.7% in cattle, 0.7% in sheep and 0.3% in pigs. A significant decrease in sheep was detected from the previous survey (1.7%). Salmonella carriage was 1.4% in cattle, a significant increase from the previous survey of 0.2%. In sheep, faecal carriage was 1.1% a significant increase from the previous survey (0.1%). In pigs, carriage was 23.4%, consistent with the previous study. Thermophilic Campylobacter spp. were isolated from 54.6% of cattle, 43.8% of sheep and 69.3% of pigs. Y. enterocolitica was isolated from 4.5% of cattle, 8.0% of sheep and 10.2% of pigs.

Molecular Typing of Salmonella Serotypes Prevalent in Animals in England: Assessment of Methodology

ABSTRACT 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. BothPstI/SphI ribotyping andXbaI-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.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations.

Longitudinal Farm Study of Extended-Spectrum β-Lactamase-Mediated Resistance

ABSTRACT Extended-spectrum β-lactamase (ESBL)-mediated resistance is of considerable importance in human medicine. Recently, such enzymes have been reported in bacteria from animals. We describe a longitudinal study of a dairy farm suffering calf scour with high mortality rates. In November 2004, two Escherichia coli isolates with resistance to a wide range of β-lactams (including amoxicillin-clavulanate and cefotaxime) were isolated from scouring calves. Testing by PCR and sequence analysis confirmed the isolates as being both blaCTX-M14/17 and blaTEM-35(IRT-4) positive. They had indistinguishable plasmid and pulsed-field gel electrophoresis (PFGE) profiles. Transferability studies demonstrated that blaCTX-M was located on a conjugative 65-MDa IncK plasmid. Following a farm visit in December 2004, 31/48 calves and 2/60 cows were positive for E. coli with blaCTX-M. Also, 5/48 calf and 28/60 cow samples yielded blaCTX- and blaTEM-negative E. coli isolates that were resistant to cefotaxime, and sequence analysis confirmed that these presented mutations in the promoter region of the chromosomal ampC gene. Fingerprinting showed 11 different PFGE types (seven in blaCTX-M-positive isolates). Six different PFGE clones conjugated the same blaCTX-M-positive IncK plasmid. One clone carried a different-sized, blaCTX-M-positive, transformable plasmid. This is the first report of blaCTX-M from livestock in the United Kingdom, and this report demonstrates the complexity of ESBL epidemiology. Results indicate that horizontal plasmid transfer between strains as well as horizontal gene transfer between plasmids have contributed to the spread of resistance. We have also shown that some clones can persist for months, suggesting that clonal spread also contributes to the perpetuation of resistance.

Epidemiology of anthroponotic and zoonotic human cryptosporidiosis in England and Wales, 2004–2006

In order to monitor epidemiological trends, Cryptosporidium-positive samples (n=4509) from diarrhoeic patients were typed. Compared to the previous 4 years, the proportion of Cryptosporidium hominis cases in 2004-2006 increased to 57·3%, while 38·5% were C. parvum. The remaining 4·2% cases included mixed C. parvum and C. hominis infections, C. meleagridis, C. felis, C. ubiquitum and a novel genotype. When the typing results were combined with enhanced surveillance data to monitor risk exposures, C. hominis was linked to urban dwelling, previous diarrhoea in the household, any travel especially abroad, and using a swimming or paddling pool. C. parvum was linked to having a private water supply, contact with surface water, visiting or living on a farm, and contact with farm animal faeces. The proportion of laboratory-confirmed indigenous cases acquired from direct contact with farm animals was estimated to be 25% for C. parvum and 10% of all reported Cryptosporidium cases.

Distribution of Cryptosporidium species in sheep in the UK

There have been few studies of the distribution of Cryptosporidium species and genotypes in sheep, and the anthropozoonotic potential has been questioned since one of the major human pathogens, Cryptosporidium parvum, is not always found. To investigate the situation in the UK we undertook three studies: a reactive sampling programme of flocks identified as exposures for human cases of cryptosporidiosis; investigation of neonatal cryptosporidiosis in lambs; and a screening programme of lambs at an open farm. C. parvum was the only species found in neonatal lambs with cryptosporidiosis and predominated in flocks sampled reactively to a human case of cryptosporidiosis. C. bovis was also found in the latter study but at a lower frequency than C. parvum. C. bovis and the cervine genotype were found in the orphan lambs under the screening programme. The results of these studies show that C. parvum is important in neonatal lamb diarrhoea and is widespread in sheep flocks in the UK, but that other Cryptosporidium species and genotypes are also present. Sheep, and young lambs in particular, must still be considered as a source of C. parvum infection for humans.

Molecular fingerprinting evidence of the contribution of wildlife vectors in the maintenance of Salmonella Enteritidis infection in layer farms

Aims: To provide molecular fingerprinting evidence of the contribution of wildlife vectors in the on‐farm epidemiology of Salmonella Enteritidis infections.

Comparison of gyrA mutations, cyclohexane resistance, and the presence of class I integrons in Salmonella enterica from farm animals in England and Wales.

ABSTRACT This study is focused on real-time detection of gyrA mutations and of the presence of class I integrons in a panel of 100 veterinary isolates of Salmonella enterica from farm animals. The isolates were selected on the basis of resistance to nalidixic acid, representing a variety of the most prevalent serotypes in England and Wales. In addition, organic solvent (cyclohexane) resistance in these isolates was investigated in an attempt to elucidate the presence of efflux pump mechanisms. The most prevalent mutation among the isolates studied was Asp87-Asn (n = 42), followed by Ser83-Phe (n = 38), Ser83-Tyr (n = 12), Asp87-Tyr (n = 4), and Asp87-Gly (n = 3). Two distinct subpopulations were identified, separated at the 1-mg/liter breakpoint for ciprofloxacin: 86% of isolates with mutations in codon 83 showed MICs of ≥1 mg/liter, while 89.8% of isolates with mutations in codon 87 presented MICs of ≤0.5 mg/liter. Cyclohexane resistance was more prevalent among Ser83 mutants than among Asp87 mutants (34.7 and 4%, respectively), and in 79% of isolates that presented both gyrA mutations and cyclohexane resistance, the level of ciprofloxacin resistance was ≥2.0 mg/liter. Thirty-four isolates contained class I integrons, with 71% of the S. enterica serovar Typhimurium isolates and 6.9% of isolates belonging to other serotypes containing such elements. The methods used represent sensitive ways of investigating the presence of gyrA mutations and of detecting class-I integrons in Salmonella isolates. The results can be obtained in less than 1 h from single colonies without the need for purifying DNA.

A laboratory study of an inactivated bivalent iron restricted Salmonella enterica serovars Enteritidis and Typhimurium dual vaccine against Typhimurium challenge in chickens

A commercial inactivated iron restricted Salmonella Typhimurium and Salmonella Enteritidis vaccine was used to vaccinate chicks at 1 day and again at 4 weeks of age, with challenge by a high and a low dose of S. Typhimurium given either orally or by contact with seeder birds inoculated orally with a high dose of S. Typhimurium. In all three challenge regimes, the shedding of challenge strain was reduced significantly (p < 0.05) in vaccinated birds compared with unvaccinated controls. Vaccination reduced colonisation of internal organs after challenge by contact seeder birds. However, no effect of vaccination upon colonisation of internal organs after either high or low oral challenge was apparent. In conclusion, the data indicate that the vaccine should be a useful tool in the control of S. Typhimurium infection in chickens.

Multiple Genetic Typing of Salmonella enterica Serotype Typhimurium Isolates of Different Phage Types (DT104, U302, DT204b, and DT49) from Animals and Humans in England, Wales, and Northern Ireland

ABSTRACT Salmonella enterica serotype Typhimurium is a common cause of salmonellosis among humans and animals in England, Wales, and Northern Ireland. Phage types DT104 and U302 were the most prevalent types in both livestock and humans in 2001. In addition, Salmonella serotype Typhimurium DT204b was responsible for a recent international outbreak involving England. A total of 119 isolates from humans (n = 28) and animals or their environment (n = 91), belonging to DT104 (n = 66), U302 (n = 33), DT204b (n = 12), and DT49 (n = 8), were fingerprinted by a combination of well-established genetic methods (pulsed-field gel electrophoresis [PFGE], PstI/SphI [PS] ribotyping, and plasmid profiling). The different techniques identified different degrees of polymorphism (from greatest to least, plasmid profiling [40 types], PS ribotyping [34 types], and PFGE [23 types]). It seems clear that a prevalent genomic clone, as well as a variety of less frequent clones, is present for each of the phage types. In most cases, the prevalent clones appeared within isolates from several animal species and from several geographical locations. We did not find clear evidence of a higher degree of diversity for any of the animal species included, or of any link between isolates from particular animal species and humans. The data presented show the inaccuracy of drawing epidemiological conclusions based on a single fingerprinting method. Strains that share one of the markers do not necessarily belong to the same clone, and a multiple typing approach is required to enable enough discrimination to track strains for epidemiological investigations.