Susanna Williamson

Colistin resistance in Salmonella and Escherichia coli isolates from a pig farm in Great Britain

OBJECTIVES The objective of this study was to characterize colistin-resistant bacteria isolated from pigs on a farm in Great Britain following identification of a plasmid-borne colistin resistance mechanism in Escherichia coli from China. METHODS Phenotypic antimicrobial susceptibility testing was undertaken by broth dilution and WGS was performed to detect the presence of genes encoding resistance and virulence. Transferable colistin resistance was investigated by conjugation. RESULTS Two E. coli and one Salmonella Typhimurium variant Copenhagen were shown to be MDR, including resistance to colistin, with one E. coli and the Salmonella carrying the mcr-1 gene; all three harboured chromosomal mutations in genes conferring colistin resistance and both E. coli harboured β-lactamase resistance. The Salmonella mcr-1 plasmid was highly similar to pHNSHP45, from China, while the E. coli mcr-1 plasmid only had the ISApII and mcr-1 genes in common. The frequency of mcr-1 plasmid transfer by conjugation to recipient Enterobacteriaceae from Salmonella was low, lying between 10(-7) and 10(-9) cfu/recipient cfu. We were unable to demonstrate mcr-1 plasmid transfer from the E. coli. Plasmid profiling indicated transfer of multiple plasmids from the Salmonella resulting in some MDR transconjugants. CONCLUSIONS Identification of the mcr-1 gene in Enterobacteriaceae from pigs confirms its presence in livestock in Great Britain. The results suggest dissemination of resistance through different horizontally transferable elements. The in vitro transfer of multiple plasmids carrying colistin and other resistances from the Salmonella isolate underlines the potential for wider dissemination and recombination.

Transplacental transmission of bluetongue virus 8 in cattle, UK.

To determine whether transplacental transmission could explain overwintering of bluetongue virus in the United Kingdom, we studied calves born to dams naturally infected during pregnancy in 2007–08. Approximately 33% were infected transplacentally; some had compromised health. In all infected calves, viral load decreased after birth; no evidence of persistent infection was found.

Risk factors for porcine reproductive and respiratory syndrome virus infection and resulting challenges for effective disease surveillance

BackgroundThis study aimed to identify risk factors for active porcine reproductive and respiratory syndrome virus (PRRSV) infection at farm level and to assess the probability of an infected farm being detected through passive disease surveillance in England. Data were obtained from a cross-sectional study on 147 farrow-to-finish farms conducted from April 2008 – April 2009. The risk factors for active PRRSV infection were identified using multivariable logistic regression analysis. The surveillance system was evaluated using a stochastic scenario tree model.ResultsEvidence of PRRSV circulation was confirmed on 35.1% (95%CI: 26.8-43.4) of farms in the cross sectional study, with a higher proportion of infected farms in areas with high pig density (more than 15000 pigs within 10 km radius from the farm). Farms were more likely to have active PRRSV infection if they used the live virus vaccine-Porcilis PRRS (OR=7.5, 95%CI: 2.5-22.8), were located in high pig density areas (OR=2.9, 95%CI: 1.0-8.3) or had dead pigs collected (OR=5.6, 95%CI: 1.7-18.3). Farms that weaned pigs at 28 days of age or later had lower odds of being PRRSV positive compared to those weaning at 21-27 days (OR=0.2, 95%CI: 0.1-0.7). The probability of detecting an infected farm through passive surveillance for disease was low (mode=0.074, 5th and 95th percentiles: 0.067; 0.083 respectively). In particular farms which used live virus vaccine had lower probabilities for detection compared to those which did not.ConclusionsRisk factors identified highlight the importance of biosecurity measures for the incursion of PRRSV infection. The results further indicate that a combined approach of surveillance for infection and disease diagnosis is needed to assist effective control and/or elimination of PRRSV from the pig population.

Porcine Epidemic Diarrhea Virus among Farmed Pigs, Ukraine

An outbreak of porcine epidemic diarrhea occurred in the summer of 2014 in Ukraine, severely affecting piglets <10 days of age; the mortality rate approached 100%. Full genome sequencing showed the virus to be closely related to strains reported from North America, showing a sequence identity of up to 99.8%.

Prevalence and risk factors for swine influenza virus infection in the English pig population.

Infection of pigs with influenza viruses is a cause of considerable economic loss for pig farmers as well as a potential human health concern - as evidenced by the identification of genetic material derived from swine-adapted influenza viruses in an novel strain of H1N1 influenza virus in 2009. A study was conducted investigating the prevalence of influenza virus infection in a selection of 143 English pig herds between April 2008 and April 2009, which found evidence of recent virus circulation in over half of these herds (n=75). Farms which were sampled in the Summer months were found to have lower odds of recent virus circulation, as were farms containing pigs kept in straw yards. Additionally, farms containing pigs kept indoors and farms containing high numbers of finisher pigs per water space were found to have higher odds of recent virus circulation. It is hoped that further studies will expand on these findings, and may allow targeting of surveillance for influenza viruses in the English pig population.

Porcine reproductive and respiratory syndrome virus: genetic diversity of recent British isolates.

Porcine reproductive and respiratory syndrome (PRRS) continues to be a significant problem for European pig producers, contributing to porcine respiratory disease complex, neonatal piglet mortality, infertility and occasional abortion storms. PRRS virus (PRRSV), a member of the arterivirus family with two defined major genotypes, has been shown to be quite genetically diverse. In the present study, genetic analysis of multiple gene regions of over 100 viruses isolated in Britain between 2003 and 2007 revealed that the diversity of British strains is now far greater than during the early 1990s. All isolates belong to genotype 1 (European). While some recent isolates are still very similar to early isolates, a wide range of more diverse viruses is now also circulating. Interestingly, some isolates were found to be very similar to a modified-live vaccine strain, and it is suggested that use of the vaccine has affected the evolution pattern of PRRS virus strains in Britain. Evidence of deletions in one viral gene, ORF3, and of genome recombination was also seen. A molecular clock model using the ORF7 sequences estimates the rate of substitution as 3.8 × 10(-3) per site per year, thereby dating the most recent common ancestor of all British viruses to 1991, coincident with the first outbreak of disease. Our findings therefore have implications for both the diagnostic and prophylactic methods currently being used, which are discussed.

Differential diagnosis of bluetongue in cattle and sheep

IN 2006, bluetongue virus serotype 8 (BTV-8) caused the first recorded bluetongue outbreak in northern Europe. This occurred in countries with no previous history of bluetongue virus incursion, including the Netherlands, Belgium and Germany. Following a halt to disease transmission over the winter, the virus re-emerged in 2007, spreading throughout northern Europe and eventually reaching the UK in September 2007. The chances that BTV-8 will emerge again in the UK in 2008 are considered to be very high. Due to the wide spectrum of clinical signs and variation in severity of disease seen in cattle and sheep with bluetongue, a variety of diseases may resemble some or all features of bluetongue infection. This article describes the main differential diagnoses of bluetongue in cattle and sheep, and summarises the distinguishing features and laboratory tests that can assist in their differentiation.

Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

ABSTRACT Haemophilus parasuis causes Glässers disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.

Multiplex PCR Assay for Unequivocal Differentiation of Actinobacillus pleuropneumoniae Serovars 1 to 3, 5 to 8, 10, and 12

ABSTRACT An improved multiplex PCR, using redesigned primers targeting the serovar 3 capsule locus, which differentiates serovars 3, 6, and 8 Actinobacillus pleuropneumoniae isolates, is described. The new primers eliminate an aberrant serovar 3-indicative amplicon found in some serovar 6 clinical isolates. Furthermore, we have developed a new multiplex PCR for the detection of serovars 1 to 3, 5 to 8, 10, and 12 along with apxIV, thus extending the utility of this diagnostic PCR to cover a broader range of isolates.

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae

Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Methods Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. Results A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. Conclusions This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial.