Design and Validation of RT-PCR Assays to Differentiate Salmonella Vaccine Strains from Wild-Type Field Isolates.

The timely differentiation of the AviPro Salmonella VAC T and VAC E strains from the wild-type Salmonella enterica ser. Typhimurium and ser. Enteritidis isolates is crucial for effectively monitoring veterinary isolates. Currently, the distinction between field and vaccine strains has been conducted routinely via phenotypic antimicrobial resistance testing since the vaccines were first introduced more than 20 years ago, and the differentiation based on the antimicrobial resistance profile is still a valid and well-established method. However, an alternative method was sought for those laboratories that prefer a PCR-based method for logistic and/or operational reasons. In this study, we developed two triplex Real-Time PCR reactions that targeted conserved and specific mutations and, therefore, enabled the reliable differentiation of field and vaccine strains. To validate the effectiveness of both assays, we extensively tested them on a dataset consisting of 405 bacterial strains. The results demonstrated a 100% sensitivity and specificity for distinguishing both Salmonella enterica ser. Typhimurium and Enteritidis, although a confirmed culture is required.

Early vaccination of laying hens with the live bivalent Salmonella vaccine AviPro™ Salmonella DUO results in successful vaccine uptake and increased gut colonization.

Introduction: Salmonella Enteritidis and S. Typhimurium are the two most clinically important zoonotic Salmonella serovars and vaccination of breeding and laying hens affords effective Salmonella control. The use of live vaccines has proven beneficial for a number of reasons, including ease of application, protection from the first day of life onwards and initiation of a strong local immune response. Live vaccines can be applied in the drinking water from the first day of life onwards, but some rearers choose to wait until the end of the first week to ensure sufficient water consumption. However, this practice leaves the birds unprotected during the crucial first week of life, where they are most susceptible to colonization by field strains. The aim of this study was to determine if successful vaccine uptake is achieved when layer pullets are vaccinated as early as day one. Methods: Three pullet flocks were vaccinated at 1, 2, 3 or 5 days-of-age with AviPro™ Salmonella DUO, a live vaccine containing attenuated strains of S. Enteritidis and S. Typhimurium (Elanco Animal Health, Cuxhaven, Germany). The vaccine was administered via the drinking water following manufacturer's instructions. Two days post-vaccination, 10 birds per flock were culled and caecal and liver samples taken, along with two pools of faeces per flock. Levels of vaccine strains were determined by quantitative and qualitative bacteriology. Results: Vaccine strains were detected in all birds from all age groups indicating successful uptake of the vaccine. Levels of the S. Enteritidis vaccine were higher than levels of the S. Typhimurium vaccine, with the latter frequently only detectable following enrichment. There was an inverse correlation between age and caecal levels of vaccines, with the highest numbers seen in birds vaccinated at 1-day-of-age. Interestingly, S. Enteritidis vaccine strain levels in liver samples were highest when birds were vaccinated at 5 days-of-age. Discussion: These results show that successful uptake of both vaccine strains was evident in all age groups. The earlier the chicks were vaccinated, the higher the vaccine levels in caecal contents. We therefore recommend vaccination of pullets as early as practicably possible to ensure protection against exposure to field strains.

Outbreak of Shiga toxin-producing Escherichia coli O157:H7 linked to raw drinking milk resolved by rapid application of advanced pathogen characterisation methods, England, August to October 2017.

An outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 occurred on the Isle of Wight between August and October 2017. Of the seven cases linked to the outbreak, five were identified through the statutory notification process and two were identified through national surveillance of whole genome sequencing data. Enhanced surveillance questionnaires established a common link to a farm, and link to the likely food vehicle, raw drinking milk (RDM). Microbiological investigations, including PCR, identified the presence of STEC O157:H7 in samples of RDM. Analysis of core genome single nucleotide polymorphism (SNP) data of STEC O157:H7 from human stool specimens, animal faecal samples and RDM demonstrated a one SNP difference between isolates, and therefore close genetic relatedness. Control measures that were put in place included suspension of sales and recall of RDM, as well as restrictions on public access to parts of the farm. Successful integration of traditional epidemiological surveillance and advanced laboratory methods for the detection and characterisation of STEC O157:H7 from human, animal and environmental samples enabled prompt identification of the outbreak vehicle and provided evidence to support the outbreak control team's decision-making, leading to implementation of effective control measures in a timely manner.

Observations on the distribution and persistence of monophasic Salmonella Typhimurium on infected pig and cattle farms.

Following a rapid rise in cases of monophasic Salmonella Typhimurium DT193 (mST) in humans and pigs since 2007 a detailed study of the prevalence and persistence of mST on pig and cattle farms in Great Britain (GB) was undertaken. Thirteen commercial pig farms and twelve cattle farms, identified as mST-positive from surveillance data, were intensively sampled over a three year period. Five indoor and eight outdoor pig farms and four beef and eight dairy farms were included. Individual and pooled faecal samples were collected from each epidemiological group and environmental samples throughout each farm and the antimicrobial resistance profile determined for a selection of mST-positive isolates. Indoor pig farms had a higher mST prevalence than outdoor pig farms, and across both cattle and pig farms the juvenile animals had a higher mST prevalence than the adult animals. Overall, mST prevalence decreased with time across all pig farms, from 25% to less than 15% of environmental samples and 22% to 15% of pooled faecal samples; only one organic outdoor breeding farm was Salmonella-negative at the end of the study. Across the cattle farms no mST was detected by the end of the study, apart from one persistent farm. Clearance time of mST was between seven and twenty-five months. Farms were selected based on having the antimicrobial resistance profile ampicillin, streptomycin, sulphonamides and tetracycline (A, S, SU, T), although resistance to trimethoprim-potentiated sulphamethoxazole was also identified on five pig farms sampled. This study provided a detailed insight into the distribution and persistence of mST on individual pig and cattle farms in GB. It has identified variation in mST shedding of individual animals, and the data can be applied to the wider livestock industry when considering the distribution of mST once identified on an individual farm.

Estimation of the sensitivity of various environmental sampling methods for detection of Salmonella in duck flocks.

Reports of Salmonella in ducks in the UK currently rely upon voluntary submissions from the industry, and as there is no harmonized statutory monitoring and control programme, it is difficult to compare data from different years in order to evaluate any trends in Salmonella prevalence in relation to sampling methodology. Therefore, the aim of this project was to assess the sensitivity of a selection of environmental sampling methods, including the sampling of faeces, dust and water troughs or bowls for the detection of Salmonella in duck flocks, and a range of sampling methods were applied to 67 duck flocks. Bayesian methods in the absence of a gold standard were used to provide estimates of the sensitivity of each of the sampling methods relative to the within-flock prevalence. There was a large influence of the within-flock prevalence on the sensitivity of all sample types, with sensitivity reducing as the within-flock prevalence reduced. Boot swabs (individual and pool of four), swabs of faecally contaminated areas and whole house hand-held fabric swabs showed the overall highest sensitivity for low-prevalence flocks and are recommended for use to detect Salmonella in duck flocks. The sample type with the highest proportion positive was a pool of four hair nets used as boot swabs, but this was not the most sensitive sample for low-prevalence flocks. All the environmental sampling types (faeces swabs, litter pinches, drag swabs, water trough samples and dust) had higher sensitivity than individual faeces sampling. None of the methods consistently identified all the positive flocks, and at least 10 samples would be required for even the most sensitive method (pool of four boot swabs) to detect a 5% prevalence. The sampling of dust had a low sensitivity and is not recommended for ducks.

A study of the dynamics of Salmonella infection in turkey breeding, rearing and finishing houses with special reference to elimination, persistence and introduction of Salmonella.

In this descriptive study, the dynamics of Salmonella infection of turkey flocks were investigated by repeated sampling of houses where Salmonella had been identified. The aim of the study was to identify the most common scenarios involved in elimination, persistence and introduction of Salmonella in the different branches of the turkey industry. Sixty-two houses on 34 turkey farms (comprising breeding, rearing and finishing farms) were sampled longitudinally, starting with the identification of a positive flock. A total of 117 follow-on flocks were tested and cleaning and disinfection (C&D) was assessed during 66 post-C&D visits. A total of 155 incidents (clearance, persistence or introduction of Salmonella) were recorded. Persistence was seen in 35.5% of incidents and was seen more frequently in breeding and rearing houses compared with finishing houses. Most persistence incidents were the result of insufficient C&D. Clearance was seen in 40% of incidents and was more often observed in finishing houses than in breeding or rearing houses. Introduction was seen in 24.5% of incidents and was more common in breeding and finishing flocks than in rearing flocks. Contamination of a house with Salmonella Typhimurium was more likely to be cleared compared with other serovars. The total number of positive samples found at a post-C&D visit was correlated with the probability of carry-over of infection, whereas the location of the positive samples seemed to be less important. Our highly sensitive post-C&D sampling method allowed us to predict a negative follow-on flock in most cases.

A retrospective analysis of Salmonella serovars isolated from pigs in Great Britain between 1994 and 2010.

A retrospective study analysing Salmonella serovars and antimicrobial resistance data from pigs in Great Britain between 1994 and 2010 was performed. Data were obtained through scanning surveillance and compared with prevalence data from other livestock species as well as human data. During the study period, two serovars, S. Typhimurium and S. Derby, predominated in British pigs. The total number of Salmonella incidents has decreased steadily over the study period, from 360 incidents per year in 1994 to 172 incidents in 2010. S. Typhimurium has been the most common serovar every year, but the relative percentage went down over the past few years. During the same time period, monophasic strains of S. Typhimurium have increased in numbers and were accounting for as much as 25% of incidents in 2010, representing the second most common serovar in British pigs in 2010. Antimicrobial resistance data from S. Typhimurium isolates show that the percentage of isolates which are resistant to six or more antimicrobials has increased from 27.2% in 1994 to 58.3% in 2010 and that the percentage of isolates fully sensitive to the panel of antimicrobials tested was only 3.3% in 2010. S. Typhimurium isolates belonged mainly to phagetypes DT193 and U288 in 2010, and an increase can be seen in the number of isolates belonging to phage type DT193. DT104, which was the predominant phagetype during the 1990s, has gone down considerably and represented less than 5% of all S. Typhimurium isolates in 2010. Monophasic strains of S. Typhimurium belonged mainly to phagetype 193 with less than 20% belonging to phagetype 120. The overall trend shows that the number of Salmonella incidents reported from British pigs has gone down considerably over the 17-year study period, but that the relative percentage of monophasic strains of S. Typhimurium has increased significantly. This trend seems to be in line with observations from other European countries, where an increase of this serovar can be seen both in livestock and in human patients. The increasing percentage of Salmonella-isolates which show multi-resistance is a matter of concern and has to be monitored carefully in order to assess any potential risks this may pose to human patients.

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.