Veterinary Medicines Directorate

Foot-and-mouth is one of the world's most economically important livestock diseases. We developed an individual farm-based stochastic model of the current UK epidemic. The fine grain of the epidemiological data reveals the infection dynamics at an unusually high spatiotemporal resolution. We show that the spatial distribution, size, and species composition of farms all influence the observed pattern and regional variability of outbreaks. The other key dynamical component is long-tailed stochastic dispersal of infection, combining frequent local movements with occasional long jumps. We assess the history and possible duration of the epidemic, the performance of control strategies, and general implications for disease dynamics in space and time.

Parabens are used as preservatives in many thousands of cosmetic, food and pharmaceutical products to which the human population is exposed. Although recent reports of the oestrogenic properties of parabens have challenged current concepts of their toxicity in these consumer products, the question remains as to whether any of the parabens can accumulate intact in the body from the long-term, low-dose levels to which humans are exposed. Initial studies reported here show that parabens can be extracted from human breast tissue and detected by thin-layer chromatography. More detailed studies enabled identification and measurement of mean concentrations of individual parabens in samples of 20 human breast tumours by high-pressure liquid chromatography followed by tandem mass spectrometry. The mean concentration of parabens in these 20 human breast tumours was found to be 20.6 +/- 4.2 ng x g(-1) tissue. Comparison of individual parabens showed that methylparaben was present at the highest level (with a mean value of 12.8 +/- 2.2 ng x g(-1) tissue) and represents 62% of the total paraben recovered in the extractions. These studies demonstrate that parabens can be found intact in the human breast and this should open the way technically for more detailed information to be obtained on body burdens of parabens and in particular whether body burdens are different in cancer from those in normal tissues.

Newcastle disease (ND), caused by avian paramyxovirus serotype 1 (APMV-1) viruses, is included in List A of the Office International des Epizooties. Historically, ND has been a devastating disease of poultry, and in many countries the disease remains one of the major problems affecting existing or developing poultry industries. Even in countries where ND may be considered to be controlled, an economic burden is still associated with vaccination and/or maintaining strict biosecurity measures. The variable nature of Newcastle disease virus strains in terms of virulence for poultry and the different susceptibilities of the different species of birds mean that for control and trade purposes, ND requires careful definition. Confirmatory diagnosis of ND requires the isolation and characterisation of the virus involved. Assessments of virulence conventionally require in vivo testing. However, in vitro genetic characterisation of viruses is being used increasingly now that the molecular basis of pathogenicity is more fully understood. Control of ND is by prevention of introduction and spread, good biosecurity practices and/or vaccination. Newcastle disease viruses may infect humans, usually causing transient conjunctivitis, but human-to-human spread has never been reported. Eight other serotypes of avian paramyxoviruses are recognised, namely: APMV-2 to APMV-9. Most of these serotypes appear to be present in natural reservoirs of specific feral avian species, although other host species are usually susceptible. Only APMV-2 and APMV-3 viruses have made a significant disease and economic impact on poultry production. Both types of viruses cause respiratory disease and egg production losses which may be severe when exacerbated by other infections or environmental stresses. No reports exist of natural infections of chickens with APMV-3 viruses.

Background: Only recently has the environment been clearly implicated in the risk of antibiotic resistance to clinical outcome, but to date there have been few documented approaches to formally assess these risks.Objective: We examined possible approaches and sought to identify research needs to enable human health risk assessments (HHRA) that focus on the role of the environment in the failure of antibiotic treatment caused by antibiotic-resistant pathogens.Methods: The authors participated in a workshop held 4–8 March 2012 in Québec, Canada, to define the scope and objectives of an environmental assessment of antibiotic-resistance risks to human health. We focused on key elements of environmental-resistance-development “hot spots,” exposure assessment (unrelated to food), and dose response to characterize risks that may improve antibiotic-resistance management options.Discussion: Various novel aspects to traditional risk assessments were identified to enable an assessment of environmental antibiotic resistance. These include a) accounting for an added selective pressure on the environmental resistome that, over time, allows for development of antibiotic-resistant bacteria (ARB); b) identifying and describing rates of horizontal gene transfer (HGT) in the relevant environmental “hot spot” compartments; and c) modifying traditional dose–response approaches to address doses of ARB for various health outcomes and pathways.Conclusions: We propose that environmental aspects of antibiotic-resistance development be included in the processes of any HHRA addressing ARB. Because of limited available data, a multicriteria decision analysis approach would be a useful way to undertake an HHRA of environmental antibiotic resistance that informs risk managers.Citation: Ashbolt NJ, Amézquita A, Backhaus T, Borriello P, Brandt KK, Collignon P, Coors A, Finley R, Gaze WH, Heberer T, Lawrence JR, Larsson DG, McEwen SA, Ryan JJ, Schönfeld J, Silley P, Snape JR, Van den Eede C, Topp E. 2013. Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance. Environ Health Perspect 121:993–1001; http://dx.doi.org/10.1289/ehp.1206316

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette-Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding sigma-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

AIMS: This study was designed to compare the degree of inhibition of platelet aggregation (IPA) of prasugrel with that of clopidogrel in stable aspirin-treated patients with coronary artery disease (CAD). METHODS AND RESULTS: Subjects (n=101) were randomly assigned to the following loading dose (LD) (day 1)/maintenance dose (MD) (days 2-28) combinations: prasugrel, 40 mg/5 mg; 40 mg/7.5 mg; 60 mg/10 mg; 60 mg/15 mg; or clopidogrel, 300 mg/75 mg. Turbidometric platelet aggregation was measured at multiple timepoints during the study. At 4 h after dosing, with 20 microM ADP, both prasugrel LDs achieved significantly higher mean IPA levels (60.6% and 68.4 vs. 30.0%, respectively; all P<0.0001) and lower percentage (3 vs. 52%, P<0.0001) of pharmacodynamic non-responders (defined as IPA <20%) than clopidogrel. Prasugrel 10 and 15 mg MDs achieved consistently higher mean IPA than clopidogrel 75 mg at day 28 (all P<0.0001). At pre-MD on day 28, there were no non-responders in the 10 and 15 mg prasugrel group, compared with 45% in the clopidogrel group (P=0.0007). CONCLUSION: In this population, prasugrel (40-60 mg LD and 10-15 mg MD) achieves greater IPA and a lower proportion of pharmacodynamic non-responders compared with the approved clopidogrel dosing.

method. The major disadvantages of both of these techniques are the high number of untypeable strains and the time-consuming and technically demanding requirements of the techniques. Production and quality control of antiserum reagents for serotyping schemes are costly; consequently, these reagents are not widely available. A recently developed scheme (23) based on HS antigens in which modified antibody production and antigen detection techniques are used may be an improvement for routine use, but this scheme does not solve the problem of restricted reagent availability or the problem of the high level of nontypeability. Because of such problems, the value of serotyping techniques for national and global epidemiological studies has been restricted. Thus, there is a well-recognized need for alternative subtyping schemes. Recently, molecular subtyping methods have been developed. The major advantage of genotyping techniques is that potentially they could be universally available. Some of these techniques, like ribotyping, pulsed-field gel electrophoresis (PFGE), and flagellin typing (fla typing), are already in use in a number of laboratories (62). In this review we describe the technologies currently available for genotypic subtyping of Campylobacter spp., discuss the advantages and problems of each technique, and indicate the current value of each method.

Antimicrobials are important tools for the therapy of infectious bacterial diseases in companion animals. Loss of efficacy of antimicrobial substances can seriously compromise animal health and welfare. A need for the development of new antimicrobials for the therapy of multiresistant infections, particularly those caused by Gram-negative bacteria, has been acknowledged in human medicine and a future corresponding need in veterinary medicine is expected. A unique aspect related to antimicrobial resistance and risk of resistance transfer in companion animals is their close contact with humans. This creates opportunities for interspecies transmission of resistant bacteria. Yet, the current knowledge of this field is limited and no risk assessment is performed when approving new veterinary antimicrobials. The objective of this review is to summarize the current knowledge on the use and indications for antimicrobials in companion animals, drug-resistant bacteria of concern among companion animals, risk factors for colonization of companion animals with resistant bacteria and transmission of antimicrobial resistance (bacteria and/or resistance determinants) between animals and humans. The major antimicrobial resistance microbiological hazards originating from companion animals that directly or indirectly may cause adverse health effects in humans are MRSA, methicillin-resistant Staphylococcus pseudintermedius, VRE, ESBL- or carbapenemase-producing Enterobacteriaceae and Gram-negative bacteria. In the face of the previously recognized microbiological hazards, a risk assessment tool could be applied in applications for marketing authorization for medicinal products for companion animals. This would allow the approval of new veterinary medicinal antimicrobials for which risk levels are estimated as acceptable for public health.

In February 2001, foot-and-mouth disease (FMD) was confirmed in Great Britain. A major epidemic developed, which peaked around 50 cases a day in late March, declining to under 10 a day by May. By mid-July, 1849 cases had been detected. The main control measures employed were livestock movement restrictions and the rapid slaughter of infected and exposed livestock. The first detected case was in south-east England; infection was traced to a farm in north-east England to which all other cases were linked. The epidemic was large as a result of a combination of events, including a delay in the diagnosis of the index case, the movement of infected sheep to market before FMD was first diagnosed, and the time of year. Virus was introduced at a time when there were many sheep movements around the country and weather conditions supported survival of the virus. The consequence was multiple, effectively primary, introductions of FMD virus into major sheep-keeping areas. Subsequent local spread from these introductions accounted for the majority of cases. The largest local epidemics were in areas with dense sheep populations and livestock dealers who were active during the key period. Most affected farms kept both sheep and cattle. At the time of writing the epidemic was still ongoing; however, this paper provides a basis for scientific discussion of the first five months.

The Heads of Medicines Agencies and the Federation of Veterinarians of Europe undertook a survey to gain an insight into European prescribing of antibiotics for animals, in particular to highlight the diseases for which antibiotics are most commonly said to be prescribed and which different classes, including human critically important antibiotics (CIAs). The survey was completed by 3004 practitioners from 25 European countries. Many older antibiotics (eg, penicillins, tetracyclines) are cited most frequently as the prescribed classes to treat the main food producing species. The frequency of citation of non-CIAs predominates. CIAs are mostly frequently cited to be prescribed for: urinary diseases in cats (62 per cent), respiratory diseases in cattle (45 per cent), diarrhoea in cattle and pigs (respectively 29 per cent and 34 per cent), locomotion disorders in cattle (31 per cent), postpartum dysgalactia syndrome complex in pigs (31 per cent) and dental disease in dogs (36 per cent). Clear 'preferences' between countries can be observed between antibiotic classes. The use of national formularies and guidance helps to drive responsible use of antibiotics and can significantly reduce the extent of use of CIAs. A more widespread introduction of veterinary practice antibiotic prescribing policies and monitoring obedience to these should ensure more widespread compliance with responsible use guidelines.

A sequence 375 nucleotides in length, which included the region encoding the cleavage activation site and signal peptide of the fusion protein gene, was determined for 174 isolates of Newcastle disease virus (avian paramyxovirus type 1). These were compared with the sequences of 164 isolates published on GenBank, and the resulting alignment was analysed phylogenetically using maximum likelihood. The results are presented as unrooted phylogenetic trees. Briefly, the isolates divided into six broadly distinct groups (lineages 1 to 6). Lineages 3 and 4 were further subdivided into four sublineages (a to d) and lineage 5 into five lineages (a to e). Considerable genetic heterogeneity was detected within avian paramyxoviruses type 1, which appears to be influenced by host, time and geographical origin. It is concluded that by using this dataset it will be possible to type future virus isolates rapidly on the basis of their nucleotide sequence and make inferences about their origins.

Vaccine development and the understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by the definition of immunological correlates of protection and/or pathology. To address these questions, cattle were vaccinated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and were then challenged with virulent M. bovis. Applying a semiquantitative pathology-scoring system, we were able to demonstrate that BCG vaccination imparted significant protection by reducing the disease severity on average by 75%. Analysis of cellular immune responses following M. bovis challenge demonstrated that proliferative T-cell and gamma interferon (IFN-gamma) responses towards the M. bovis-specific antigen ESAT-6, whose gene is absent from BCG, were generally low in vaccinated animals but were high in all nonvaccinated calves. Importantly, the amount of ESAT-6-specific IFN-gamma measured by enzyme-linked immunosorbent assay after M. bovis challenge, but not the frequency of responding cells, correlated positively with the degree of pathology found 18 weeks after infection. Diagnostic reagents based on antigens not present in BCG, like ESAT-6 and CFP-10, were still able to distinguish BCG-vaccinated, diseased animals from BCG-vaccinated animals without signs of disease. In summary, our results suggest that the determination of ESAT-6-specific IFN-gamma, while not a direct correlate of protection, constitutes nevertheless a useful prognostic immunological marker predicting both vaccine efficacy and disease severity.

Further preliminary observations are reported of an experiment to examine the spread of infectivity and the occurrence of pathological changes in cattle exposed orally to infection with bovine spongiform encephalopathy. Calves were dosed at four months of age and clinically monitored groups were killed sequentially from two to 40 months after inoculation. Tissues were collected for bioassay, for histopathological examinations and for the detection of PrP. Previous reported observations have included the presence of infectivity in the distal ileum of cattle killed after six to 18 months, the earliest onset of clinical signs in an exposed animal after 35 months, and diagnostic histopathological changes in the brain, in association with clinical disease, after 36, 38 and 40 months. In spite of the relative inefficiency of the bioassay of scrapie-like agents across a species barrier the new observations confirm that the onset of clinical signs and pathological changes in the central nervous system (CNS) occur at approximately the same time. The earliest pathological change, the presence of abnormal PrP 32 months after inoculation, coincided with the earliest detected infectivity in the CNS and occurred shortly before there was evidence of typical spongiform changes in the brain 36 months after inoculation. Infectivity has now been demonstrated in the peripheral nervous system, in the cervical and thoracic dorsal root ganglia 32 to 40 months after inoculation and in the trigeminal ganglion 36 and 38 months after inoculation. At the time of writing evidence of infectivity in other tissues is confined to the distal ileum, not only after six to 18 months but also after 38 and 40 months, but these findings may be supplemented by the results of further mouse assays. Nevertheless, they are in general agreement with current knowledge of the pathogenesis of scrapie.

Influenza A viruses occur worldwide in wild birds and are occasionally associated with outbreaks in commercial chickens and turkeys. However, avian influenza viruses have not been isolated from wild birds or poultry in South America. A recent outbreak in chickens of H7N3 low pathogenic avian influenza (LPAI) occurred in Chile. One month later, after a sudden increase in deaths, H7N3 highly pathogenic avian influenza (HPAI) virus was isolated. Sequence analysis of all eight genes of the LPAI virus and the HPAI viruses showed minor differences between the viruses except at the hemagglutinin (HA) cleavage site. The LPAI virus had a cleavage site similar to other low pathogenic H7 viruses, but the HPAI isolates had a 30-nucleotide insert. The insertion likely occurred by recombination between the HA and nucleoprotein genes of the LPAI virus, resulting in a virulence shift. Sequence comparison of all eight gene segments showed the Chilean viruses were also distinct from all other avian influenza viruses and represent a distinct South American clade.

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment.

A Gram-negative, non-motile, non-spore-forming coccoid bacterium (strain BO1(T)) was isolated recently from a breast implant infection of a 71-year-old female patient with clinical signs of brucellosis. Affiliation of strain BO1(T) to the genus Brucella was confirmed by means of polyamine pattern, polar lipid profile, fatty acid profile, quinone system, DNA-DNA hybridization studies and by insertion sequence 711 (IS711)-specific PCR. Strain BO1(T) harboured four to five copies of the Brucella-specific insertion element IS 711, displaying a unique banding pattern, and exhibited a unique 16S rRNA gene sequence and also grouped separately in multilocus sequence typing analysis. Strain BO1(T) reacted with Brucella M-monospecific antiserum. Incomplete lysis was detected with bacteriophages Tb (Tbilisi), F1 and F25. Biochemical profiling revealed a high degree of enzymic activity and metabolic capabilities. In multilocus VNTR (variable-number tandem-repeat) analysis, strain BO1(T) showed a very distinctive profile and clustered with the other 'exotic' Brucella strains, including strains isolated from marine mammals, and Brucella microti, Brucella suis biovar 5 and Brucella neotomae. Comparative omp2a and omp2b gene sequence analysis revealed the most divergent omp2 sequences identified to date for a Brucella strain. The recA gene sequence of strain BO1(T) differed in seven nucleotides from the Brucella recA consensus sequence. Using the Brucella species-specific multiplex PCR assay, strain BO1(T) displayed a unique banding pattern not observed in other Brucella species. From the phenotypic and molecular analysis it became evident that strain BO1( T) was clearly different from all other Brucella species, and therefore represents a novel species within the genus Brucella. Because of its unexpected isolation, the name Brucella inopinata with the type strain BO1(T) (=BCCN 09-01(T)=CPAM 6436(T)) is proposed.

The proteins ESAT-6 and CFP-10 have been shown to be secreted by Mycobacterium tuberculosis and Mycobacterium bovis cells, to be potent T-cell antigens, and to have a clear but as yet undefined role in tuberculosis pathogenesis. We have successfully overexpressed both ESAT-6 and CFP-10 in Escherichia coli and developed efficient purification schemes. Under in vivo-like conditions, a combination of fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy have shown that ESAT-6 contains up to 75% helical secondary structure, but little if any stable tertiary structure, and exists in a molten globule-like state. In contrast, CFP-10 was found to form an unstructured, random coil polypeptide. An exciting discovery was that ESAT-6 and CFP-10 form a tight, 1:1 complex, in which both proteins adopt a fully folded structure, with about two-thirds of the backbone in a regular helical conformation. This clearly suggests that ESAT-6 and CFP-10 are active as the complex and raises the interesting question of whether other ESAT-6/CFP-10 family proteins (22 paired genes in M. tuberculosis) also form tight, 1:1 complexes, and if so, is this limited to their genome partner, or is there scope for wider interactions within the protein family, which could provide greater functional flexibility?

Existing strategies for long-term bovine tuberculosis (bTB) control/eradication campaigns are being reconsidered in many countries because of the development of new testing technologies, increased global trade, continued struggle with wildlife reservoirs of bTB, redistribution of international trading partners/agreements, and emerging financial and animal welfare constraints on herd depopulation. Changes under consideration or newly implemented include additional control measures to limit risks with imported animals, enhanced programs to mitigate wildlife reservoir risks, re-evaluation of options to manage bTB-affected herds/regions, modernization of regulatory framework(s) to re-focus control efforts, and consideration of emerging testing technologies (i.e. improved or new tests) for use in bTB control/eradication programs. Traditional slaughter surveillance and test/removal strategies will likely be augmented by incorporation of new technologies and more targeted control efforts. The present review provides an overview of current and emerging bTB testing strategies/tools and a vision for incorporation of emerging technologies into the current control/eradication programs.

A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93% (56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h.

AIMS: To report the natural history of autochthonous hepatitis E and hepatitis E virus (HEV) IgG seroprevalence in Southwest England. METHODS: Patients with unexplained hepatitis were tested for hepatitis E and cases followed until recovery or death. Five hundred blood donors, 336 individuals over the age of 60 years and 126 patients with chronic liver disease were tested for HEV IgG. RESULTS: Forty cases of autochthonous hepatitis E (genotype 3) were identified. Hepatitis E was anicteric in 25% of cases and usually caused a self-limiting hepatitis predominantly in elderly Caucasian males. Six of 40 had a significant complication and three patients died, two of who had previously undiagnosed cirrhosis. Hepatitis E shows a seasonal variation with peaks in the spring and summer and no cases in November and December. HEV IgG prevalence increases with age, is more common in men and is 16% in blood donors, 13% in patients with chronic liver disease and 25% in individuals over 60 years. CONCLUSION: Autochthonous hepatitis E is more common than previously recognized, and should be considered in the differential diagnosis in patients with hepatitis, whatever their age or travel history. It carries a significant morbidity and when seen in the context of chronic liver disease carries an adverse prognosis.