Genetic Diversity of Campylobacter jejuni and Campylobacter coli Isolates from Conventional Broiler Flocks and the Impacts of Sampling Strategy and Laboratory Method.

The genetic diversity of Campylobacter jejuni and Campylobacter coliisolates from commercial broiler farms was examined by multilocus sequence typing (MLST), with an assessment of the impact of the sample type and laboratory method on the genotypes of Campylobacter isolated. A total of 645C. jejuniand 106C. coli isolates were obtained from 32 flocks and 17 farms, with 47 sequence types (STs) identified. The Campylobacter jejuniisolates obtained by different sampling approaches and laboratory methods were very similar, with the same STs identified at similar frequencies, and had no major effect on the genetic profile of Campylobacter population in broiler flocks at the farm level. ForC. coli, the results were more equivocal. While some STs were widely distributed within and among farms and flocks, analysis of molecular variance (AMOVA) revealed a high degree of genetic diversity among farms forC. jejuni, where farm effects accounted for 70.5% of variance, and among flocks from the same farm (9.9% of variance for C. jejuni and 64.1% forC. coli). These results show the complexity of the population structure of Campylobacterin broiler production and that commercial broiler farms provide an ecological niche for a wide diversity of genotypes. The genetic diversity of C. jejuni isolates among broiler farms should be taken into account when designing studies to understand Campylobacter populations in broiler production and the impact of interventions. We provide evidence that supports synthesis of studies on C. jejuni populations even when laboratory and sampling methods are not identical.

Bayesian analysis of culture and PCR methods for detection of Campylobacter spp. in broiler caecal samples.

The objective of this study was to estimate the sensitivity and specificity of a culture method and a polymerase chain reaction (PCR) method for detection of two Campylobacter species: C. jejuni and C. coli. Data were collected during a 3-year survey of UK broiler flocks, and consisted of parallel sampling of caeca from 436 batches of birds by both PCR and culture. Batches were stratified by season (summer/non-summer) and whether they were the first depopulation of the flock, resulting in four sub-populations. A Bayesian approach in the absence of a gold standard was adopted, and the sensitivity and specificity of the PCR and culture for each Campylobacter subtype was estimated, along with the true C. jejuni and C. coli prevalence in each sub-population. Results indicated that the sensitivity of the culture method was higher than that of PCR in detecting both species when the samples were derived from populations infected with at most one species of Campylobacter. However, from a mixed population, the sensitivity of culture for detecting both C. jejuni or C. coli is reduced while PCR is potentially able to detect both species, although the total probability of correctly identifying at least one species by PCR is similar to that of the culture method.

The prevalence of Campylobacter spp. in broiler flocks and on broiler carcases, and the risks associated with highly contaminated carcases.

A baseline survey on the prevalence of Campylobacter spp. in broiler flocks and Campylobacter spp. on broiler carcases in the UK was performed in 2008 in accordance with Commission Decision 2007/516/EC. Pooled caecal contents from each randomly selected slaughter batch, and neck and breast skin from a single carcase were examined for Campylobacter spp. The prevalence of Campylobacter in the caeca of broiler batches was 75·8% (303/400) compared to 87·3% (349/400) on broiler carcases. Overall, 27·3% of the carcases were found to be highly contaminated with Campylobacter (≥1000 c.f.u./g). Slaughter in the summer months (June, July, August) [odds ratio (OR) 3·50], previous partial depopulation of the flock (OR 3·37), and an increased mortality at 14 days (≥1·25% to <1·75%) (OR 2·54) were identified as significant risk factors for the most heavily Campylobacter-contaminated carcases. Four poultry companies and farm location were also found to be significantly associated with highly contaminated carcases.

Investigation of prevalence and risk factors for Campylobacter in broiler flocks at slaughter: results from a UK survey.

During 2007-2009 a UK-wide, 3-year stratified randomized survey of UK chicken broiler flocks was conducted to estimate the prevalence of Campylobacter-infected batches of birds at slaughter. Thirty-seven abattoirs, processing 88·3% of the total UK slaughter throughput, were recruited at the beginning of the survey. Of the 1174 slaughter batches sampled, 79·2% were found to be colonized with Campylobacter, the majority of isolates being C. jejuni. Previous partial depopulation of the flock [odds ratio (OR) 5·21], slaughter in the summer months (categorized as June, July and August; OR 14·27) or autumn months (categorized as September, October and November; OR 1·70) increasing bird age (40-41 days, OR 3·18; 42-45 days, OR 3·56; ⩾46 days, OR 13·43) and higher recent mortality level in the flock (1·00-1·49% mortality, OR 1·57; ⩾1·49% mortality, OR 2·74) were all identified as significant risk factors for Campylobacter colonization of the birds at slaughter. Time in transit to the slaughterhouse of more than 2·5 h was identified as a protective factor (OR 0·52).

Prevalence of Escherichia coli carrying extended-spectrum ß-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009.

OBJECTIVES: to determine the prevalence of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli from poultry in Great Britain (GB). METHODS: E. coli was isolated from 388 broiler chicken caecal samples from 22 abattoirs and from boot swabs from 442 turkey flocks over successive 1 year periods. CHROMagar ECC with and without cephalosporin antibiotics was used as isolation medium and the chicken study also used CHROMagar CTX. ESBL phenotype isolates were tested for the presence of bla(CTX-M,) bla(OXA), bla(SHV), bla(TEM) and ampC genes(.) CTX-M isolates were tested for O25 serogroup, replicon, CTX-M sequence, multilocus sequence type (MLST), PFGE type, plasmid transfer and qnrA, qnrB, qnrS, qepA and aac(6')-Ib genes. RESULTS: CTX-M-carrying E. coli were isolated from 54.5% of the broiler abattoirs and from 3.6% of individual broiler caecal samples and were CTX-M sequence types 1 (mainly), 3 and 15 with replicon types I1-γ, A/C and P/F, and I1-γ, respectively. CTX-M-carrying E. coli were isolated from 5.2% of turkey meat production farms and 6.9% of turkey breeder farms and were CTX-M sequence types 1, 14 (mainly), 15 and 55 with mainly replicon types F, FIA, K and I1-γ, respectively. None of the CTX-M isolates was serogroup O25. PFGE/MLST showed the CTX-M isolates to be clonally diverse, although MLST 156 with CTX-M-15 was isolated from both chickens and turkeys and has been previously reported in gulls. CTX-M-negative, ESBL- and bla(TEM)-positive strains were mainly TEM-52C. CONCLUSIONS: poultry-derived CTX-M E. coli in GB are different from major CTX-M sequence types causing disease in humans.

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.

Salmonella Typhimurium and Salmonella Enteritidis in England: costs to patients, their families, and primary and community health services of the NHS.

This is the first study comparing societal costs of acute illness with Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE) in the UK. It included the cost and severity of the illness and explored the impact of each Salmonella serovar on the patients, their families, the NHS, and the wider economy. The study ascertained confirmed cases of ST and SE between July and November 2008. The mean costs per case were £1282 (ST) and £993 (SE). The indirect costs associated with the work-time lost by the case, parents, or carers were £409 (ST) and £228 (SE); this difference was statistically significant. The aggregate cost of ST and SE identified using laboratory test results for the UK as a whole was estimated as £6.5 million. Work-time lost and caring activities are cost categories that are not frequently investigated within the infectious intestinal disease literature, although they represent an important societal cost.

Application of variable number of tandem repeat analysis to track Salmonella enterica ssp. enterica serovar Typhimurium infection of pigs reared on three British farms through the production cycle to the abattoir.

AIMS: This study investigated the diversity and persistence of Salmonella strains through the pork finishing cycle, from the farm into the abattoir. METHODS AND RESULTS: Isolates from four batches of finishers, from farm to abattoir, were used. Salmonella Typhimurium isolates were subjected to molecular typing using pulsed-field gel electrophoresis and variable number of tandem repeat analysis. The results demonstrated that infection was transferred from the farm to the abattoir. Within the abattoir, infection from individual pigs contaminated the exterior of the carcass and pigs exposed to Salmonella in the lairage were infected. CONCLUSIONS: Salmonella can be introduced at various points in the pig production and slaughter process. Carcass contamination may arise from infection on farm and exposure in the lairage and abattoir environment. Pigs could be contaminated by previous batches of pigs while in lairage or during the dressing process. Salmonella infection on farms is dynamic with multiple serovars present from different sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular typing methods facilitated the tracing of Salm. Typhimurium through the production cycle and differentiated some farm-acquired from abattoir-acquired strains. The findings emphasize the importance of integrated control strategies along the pork food chain.

An evaluation of survival and detection of Campylobacter jejuni and C. coli in broiler caecal contents using culture-based methods.

AIMS: To evaluate the culture specifications of the 2008 EU baseline survey for Campylobacter spp. in broiler flocks at slaughter, by assessing the detection of thermophilic Campylobacter in chicken caecal contents by culture on selective agar with or without enrichment culture. Additionally, to assess the impact of sample storage time on Campylobacter detection. METHODS AND RESULTS: Serial dilutions of pooled caeca samples in phosphate-buffered saline or Campylobacter-negative caecal contents were cultured micro-aerobically at 41.5°C on mCCDA, Karmali and Preston agars before and after enrichment in Exeter broth. Direct culture on mCCDA showed a higher isolation rate than for Karmali or Preston agars, but a similar isolation rate to enrichment. Enumeration of samples showed the numbers of viable bacteria dropped slightly during storage. CONCLUSIONS: Direct culture on mCCDA was the most sensitive method for detection of Campylobacter, and samples with 10(4) CFU g(-1) were still detectable after 6 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of prevalence results from the 2008 EU baseline survey will need careful interpretation as the different media specified vary in their sensitivity to detect thermophilic Campylobacter. Delayed culture for up to 80 h after collection should have little impact on detection rate.

Factors influencing the presence and concentration of E. coli O157 and E. coli in farm waste on six cattle farms in North-West England.

AIMS: To investigate the factors influencing the presence and burden of Escherichia coli O157 in farm wastes. METHODS AND RESULTS: Wastes from six cattle farms were screened for the presence and concentration of E. coli O157 and E. coli on three occasions over a year and waste management data were collected. Sixty-three of 878 (7.1%) samples were positive for verocytotoxigenic Escherichia coli O157 and 664/875 (75.9%) for E. coli with detectable levels greater in fresh waste than in stored waste, pasture or dirty water. CONCLUSIONS: The turning/stirring of stored waste and the use of more than one store (allowing longer storage times) reduced the proportion of E. coli O157 positive samples. The presence of E. coli O157 significantly reduced from a high prevalence found in fresh faeces and stored waste to lower proportions in dirty water and pasture samples. Escherichia coli O157 was only detected on pasture when waste was spread from contaminated stores the day before sampling. A high prevalence of positive E. coli O157 samples were detected when cattle were re-housed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings help to support the importance of treating and storing farm waste, as well as providing evidence for the level of dilution of E. coli O157 from fresh waste to recently spread pastures.

A survey of fluoroquinolone resistance in Escherichia coli and thermophilic Campylobacter spp. on poultry and pig farms in Great Britain.

AIMS: To estimate the proportions of farms on which broilers, turkeys and pigs were shedding fluoroquinolone (FQ)-resistant Escherichia coli or Campylobacter spp. near to slaughter. METHODS AND RESULTS: Freshly voided faeces were collected on 89 poultry and 108 pig farms and cultured with media containing 1.0 mg l(-1) ciprofloxacin. Studies demonstrated the specificity of this sensitive method, and both poultry and pig sampling yielded FQ-resistant E. coli on 60% of farms. FQ-resistant Campylobacter spp. were found on around 22% of poultry and 75% of pig farms. The majority of resistant isolates of Campylobacter (89%) and E. coli (96%) tested had minimum inhibitory concentrations for ciprofloxacin of > or =8 mg l(-1). The proportion of resistant E. coli and Campylobacter organisms within samples varied widely. CONCLUSIONS: FQ resistance is commonly present among two enteric bacterial genera prevalent on pig and poultry farms, although the low proportion of resistant organisms in many cases requires a sensitive detection technique. SIGNIFICANCE AND IMPACT OF THE STUDY: FQ-resistant bacteria with zoonotic potential appear to be present on a high proportion of UK pig and poultry farms. The risk this poses to consumers relative to other causes of FQ-resistant human infections remains to be clarified.

Sensitivity of Direct Culture, Enrichment and PCR for Detection of Campylobacter jejuni and C. coli in Broiler Flocks at Slaughter.

Broiler chicken flocks are a significant source of Campylobacter jejuni and Campylobacter coli that result in the major public health problem of campylobacteriosis. Accurate estimates of the prevalence of both C. coli and C. jejuni in flocks would enhance epidemiological understanding, risk assessment and control options. This study combined results from a panel of 10 detection tests (direct culture, enrichment and PCR) on caecal samples from flocks at slaughter. A parallel interpretation approach was used to determine the presence of Campylobacter spp. and for C. jejuni and C. coli individually. The sample was considered positive if at least one method detected the target and this interpretation was taken to represent a 'proxy gold standard' for detection in the absence of a gold standard reference test. The sensitivity of each individual method to detect Campylobacter spp., C. jejuni and C. coli was then estimated relative to the proxy gold standard. Enrichment in adapted Exeter broth (deficient in polymyxin B) with a resuscitation step was 100% sensitive, whilst direct culture on modified charcoal cefoperazone deoxycholate agar (mCCDA) was highly sensitive (97.9%). Enrichment methods using Preston broth and Bolton broth were significantly less sensitive. Enrichment in Exeter broth promoted the recovery of C. jejuni, whilst enrichment in Bolton broth favoured C. coli. A RT-PCR detection test could identify 80% of flocks that were co-colonised with both species. This study found that 76.3% (n = 127) of flocks were colonised with Campylobacter spp. The majority (95.9%) of Campylobacter-positive flocks were colonised with C. jejuni; however, approximately one-third of positive flocks were simultaneously colonised with both C. jejuni and C. coli. The findings highlight the impact of different detection methodologies on the accuracy of the estimated incidence of both C. jejuni and C. coli entering the abattoir within broiler flocks and the associated public health risks.

Detection of multiple cephalosporin-resistant Escherichia coli from a cattle fecal sample in Great Britain.

We describe the isolation of multiple cephalosporin-resistant Escherichia coli from cattle feces collected from animals at slaughter in Great Britain. Six E. coli strains were isolated with distinct XbaI pulsed-field gel electrophoresis (PFGE) profiles and different mechanisms of cephalosporin resistance from a single fecal sample. Two of these strains were found to contain conjugative plasmids conferring resistance to extended-spectrum cephalosporins that were indistinguishable from each other by restriction endonuclease digestion. Sequence analysis of the plasmid-encoded ampC showed that they were identical to bla(CMY-2), previously described in multiple-drug-resistant Salmonella and E. coli from animals in other parts of the world. DNA sequence analysis of the chromosomal ampC promoter regions for three cephalosporin-resistant strains lacking CMY-2 was determined. Several mutations were detected in the isolates tested including changes at positions -42 and -32, which are known to increase promoter strength. This report represents the first isolation of E. coli containing bla(CMY-2) from cattle in Great Britain, and, also to our knowledge, the first demonstration of multiple cephalosporin-resistant strains in a single animal.

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.

Factors affecting the survival of Streptococcus suis type 2.

The survival of Streptococcus suis type 2 was assessed in experimentally inoculated faeces and dust stored at 0, 9 and 22 to 25 degrees C. The organism survived in faeces for 104 days at 0 degrees C, up to 10 days at 9 degrees C and up to eight days at 22 to 25 degrees C. It survived in dust for up to 54 days at 0 degrees C and up to 25 days at 9 degrees C but could not be isolated from dust stored at room temperature for 24 hours. The organism survived at 4 degrees C in nutrient medium for up to nine months but in distilled water for only one to two weeks. At 50 degrees C it survived in water or broth for up to two hours but at 60 degrees C it only survived for 10 minutes. The organism was rapidly inactivated by disinfectants and cleansers, commonly used on farms and in laboratories, at concentrations less than those recommended for use by the manufacturers.

Further studies on the subclinical carrier state of Streptococcus suis type 2 in pigs.

Tonsil swabs were taken from 626 pigs of all ages in four infected herds. Streptococcus suis type 2 was isolated from 106 pigs aged between three weeks and 18 months. Detectable carrier rates in different groups of pigs varied between 0 and 80 per cent and were highest in weaned pigs aged between four and 10 weeks. Deep scrape cultures carried out post mortem suggested that the actual carrier rate was higher than that detected by tonsil swabs before death. Detection was improved by using two selective media. S suis type 2 persisted in the tonsils in the presence of circulating opsonic and binding antibodies and in pigs receiving penicillin-medicated feed. It was detected in the tonsils of individuals for up to 521 days. It was rarely detected in the nose. Some carriers appeared to eliminate the infection. Weaned detectable tonsil carriers transmitted the infection to previously uninfected pigs with which they were mixed. Titres of serum binding antibodies assessed by a micro enzyme-linked immunosorbent assay were relatively low and results from infected and non-infected pigs were not always distinguishable.

Monitoring herds for Streptococcus suis type 2 by sampling tonsils of slaughter pigs.

Heads were removed soon after slaughter from the dressed carcases of 155 pigs belonging to 12 herds with a history of streptococcal meningitis and from 180 pigs from four herds believed to be free from this disease. Deep scrapings from both tonsils were sown on two selective media. Streptococcus suis type 2 was detected in a proportion of pigs from the 12 herds with a history of the disease, including three herds in which no cases were noted during the year this study was made. Pigs in six of these herds had received therapeutic levels of certain antibiotics in their feed as growers. The confirmed detectable tonsillar carrier rates varied between these 12 herds from 20 to 90 per cent and between batches of heads from one herd from 0 to 100 per cent. Carrier rates could not be correlated with disease levels, herd size or husbandry system. S suis type 2 was also detected in pigs from two herds thought to be free of the disease, at rates of 20 and 1.5 per cent.

Studies of Streptococcus suis type 2 infection in pigs.

Investigations into the immunology, pathogenesis and epidemiology of Streptococcus suis type 2 infections were carried out in experimental pigs and in naturally-occurring field outbreaks of disease. The capsular polysaccharide from Str. suis type 2 was shown to induce opsonic antibodies in pigs when injected with Freund's incomplete adjuvant, but difficulties encountered in experimental production of the disease prevented a study of their protective effects. Problems with the bactericidal tests led to an investigation of other assays for antibodies against Str. suis type 2, namely, a phagocytic test with pig neutrophils, a mixed reverse passive antiglobulin haemagglutination test and an indirect haemagglutination test. There was evidence that with modifications both the latter tests would be useful. Transmission studies in 39 conventionally-reared and 7 hysterectomy-derived, colostrum-deprived pigs yielded interesting results with regard to the distribution of the organism in relation to the disease process. Tonsil carriage in clinically-healthy pigs was demonstrated after experimental and natural infection. Detectable carrier rates varied between 0 and 59%. The organism was shown to persist in the presence of circulating opsonic antibodies and in pigs on penicillin-medicated feed. Attempts to isolate the organism from the genital tract were unsuccessful. Medicated early weaning and classical SPF techniques applied to infected herds appeared to be effective in producing pigs free from Str. suis type 2 infection.

Prevalence and molecular typing of Cryptosporidium in dairy cattle in England and Wales and examination of potential on-farm transmission routes.

An average of 70 samples were collected from 80 dairy farms in England and Wales, from cattle, co-grazed sheep, wildlife and farm wastes, to investigate prevalence, potential sources and transmission routes of Cryptosporidium. At least one positive sample was detected on 74 of the farms (92.5%) by IFAT microscopy. The prevalence in cattle was 10.2% (95% CI 9.4-11.1%), with greater prevalences detected in calf samples, especially from those under 1 month (45.1%). Young calves were also more likely to be shedding Cryptosporidium parvum and larger concentrations of oocysts, whereas older calves and adult cattle were more likely to be shedding Cryptosporidium bovis and Cryptosporidium andersoni, respectively. The C. parvum subtypes detected were predominantly from types commonly identified in UK cattle (67% were either IIaA15G2R1 or IIaA17G1R1). A novel subtype, IIaA17G1R2, was identified from one cattle sample. The prevalence in co-grazed sheep was low (4%). Birds and rodents may represent significant reservoirs of Cryptosporidium due to high prevalence, large oocyst concentrations, and the detection of a C. parvum subtype known to be present in human populations, identified in samples from these wildlife. Cryptosporidium were detected in dirty water and manure, and also from pasture samples where slurry had been spread. On 64% of the farms, identical Cryptosporidium species were detected (mainly C. parvum or C. bovis) from different cattle groups on the farms, although no direct or indirect contact between the groups were recorded, apart from sharing staff. The same Cryptosporidium species were found in cattle, farm wastes and bird samples on the same farms, but rarely, or not at all, present in sheep or rodent samples. The matching of species/subtypes was also related to the proximity of the different sample sources which may indicate a potential transmission route.

A comparison between longitudinal shedding patterns of Salmonella Typhimurium and Salmonella Dublin on dairy farms.

Salmonella in cattle herds may behave as epidemic or endemic infections. An intensive longitudinal sampling study across all management groups and ages on six dairy farms in the UK was used to examine patterns of Salmonella shedding, following the prior identification of either Salmonella Dublin (SD) (three farms) or Salmonella Typhimurium (ST) (three farms) on the premises in the context of clinical salmonellosis. Individual faeces, pooled faeces and environmental samples (total 5711 samples), taken approximately every six weeks for 15-24 weeks, were cultured for Salmonella. SD was detected at low frequency (on any visit, 0.5-18.3 per cent of samples positive) and most consistently in calves. By contrast, ST was isolated at higher frequency (on any visit, 6.8-75 per cent of samples positive), and in higher numbers, up to 10(7) cfu/g faeces. Significantly more samples from calves were positive for ST than were positive for SD (50.6 per cent v 3.1 per cent; P < 0.001), which was also true for milking cows (46.3 per cent v 4.4 per cent; P < 0.001). The differences could help to explain the different patterns of bovine infection classically associated with these two serovars in the UK. No consistent effect upon shedding was seen among the ST-infected herds following vaccination.