The management of risk arising from the use of antimicrobial agents in veterinary medicine in EU/EEA countries - a review.

Antimicrobials are essential medicines for the treatment of many microbial infections in humans and animals. Only a small number of antimicrobial agents with new mechanisms of action have been authorized in recent years for use in either humans or animals. Antimicrobial resistance (AMR) arising from the use of antimicrobial agents in veterinary medicine is a concern for public health due to the detection of increasing levels of resistance in foodborne zoonotic bacteria, particularly gram-negative bacteria, and due to the detection of determinants of resistance such as Extended-spectrum beta-lactamases (ESBL) in bacteria from animals and in foodstuffs of animal origin. The importance and the extent of the emergence and spread of AMR from animals to humans has yet to be quantified. Likewise, the relative contribution that the use of antimicrobial agents in animals makes to the overall risk to human from AMR is currently a subject of debate that can only be resolved through further research. Nevertheless, risk managers have agreed that the impact on public health of the use of antimicrobials in animals should be minimized as far as possible and a variety of measures have been introduced by different authorities in the EU to achieve this objective. This article reviews a range of measures that have been implemented within European countries to reduce the occurrence and the risk of transmission of AMR to humans following the use of antimicrobial agents in animals and briefly describes some of the alternatives to the use of antimicrobial agents that are being developed.

Availability of vaccines against major animal diseases in the European Union.

This paper presents the results of a survey in which countries within the European Union and the European Economic Area were requested to provide information on the availability of vaccines against 47 major diseases of animals as part of the DISCONTOOLS project within the European Technology Platform for Global Animal Health. The objective of the survey was to help identify those diseases to which priority should be given by both the public and private sectors in terms of developing new tools to assist in their control. The survey also provides information on the availability of vaccines authorised at national level against the diseases concerned which may be useful in the event of a disease emergency or to enhance preparedness.

Incursions of foot-and-mouth disease virus into Europe between 1985 and 2006.

Foot-and-mouth disease (FMD) is one of the biggest threats to animal health in European countries. In the last 22 years (1985-2006), FMD has occurred 37 times in 14 European countries. Serotype O was most frequently involved in these outbreaks followed by A, C and Asia 1. Sometimes, epidemics were very limited and at other times, they were the cause of devastating economic losses. In most cases (22/37), the origin of the outbreaks could not be determined. For some of these outbreaks, however, routes of introduction and spread were identified through epidemiological inquiries. Moreover, in some cases, the origin of the virus was also traced by phylogenetic analysis of the partial or complete sequences of VP1 genes. Lessons learned from the outbreaks are still useful as most of the same risk factors persist. However, efforts made by FMD-free countries to help those where the disease is endemic are a valuable strategy for the reduction of the global risk. The present and the future potential sources of FMD infection need to be identified to best focus European efforts.

The importance of quality assurance/quality control of diagnostics to increase the confidence in global foot-and-mouth disease control.

The last decade international trade in animals and animal products was liberated and confidence in this global trade can increase only if appropriate control measures are applied. As foot-and-mouth disease (FMD) diagnostics will play an essential role in this respect, the Food and Agriculture Organization European Commission for the Control of Foot-and-Mouth Disease (EUFMD) co-ordinates, in collaboration with the European Commission, several programmes to increase the quality of FMD diagnostics. A quality assurance (QA) system is deemed essential for laboratories involved in certifying absence of FMDV or antibodies against the virus. Therefore, laboratories are encouraged to validate their diagnostic tests fully and to install a continuous quality control (QC) monitoring system. Knowledge of performance characteristics of diagnostics is essential to interpret results correctly and to calculate sample rates in regional surveillance campaigns. Different aspects of QA/QC of classical and new FMD virological and serological diagnostics are discussed in respect to the EU FMD directive (2003/85/EC). We recommended accepting trade certificates only from laboratories participating in international proficiency testing on a regular basis.

FMD vaccines: reflections on quality aspects for applicability in European disease control policy.

Most foot-and-mouth disease (FMD) vaccines used around the world are inactivated vaccines for prophylactic or emergency use, generally manufactured by the same basic methodology outlined in the OIE Manual and, for Europe, in the European Pharmacopoeia, and for the EU Member States in compliance with Directive 2001/82/EC of the European Parliament and of the Council of 6 November 2001 on the Community code relating to veterinary medicinal products as amended by Directive 2004/28/EC. Most of the requirements that apply to all immunological veterinary medicinal products apply equally to FMD vaccines. There are, however, some unique features of the disease and vaccines used against it that require a different approach to fulfil the requirements of the relevant legislation, if a vaccinate-to-live policy will be applied with 'authorized' vaccines. Several aspects of vaccine efficacy and safety are elaborated with emphasis on quality assurance/quality control (QA/QC). The purity of the vaccine in respect of the presence of non-structural protein antibodies could be checked indirectly by serology after vaccination. The viability of a vaccine bank approach was greatly aided by the principle of storing inactivated concentrated FMD viral antigen (Ag) over liquid nitrogen for subsequent formulation into vaccine. A worldwide Ag bank network might be an option for the far future and a solution to the problem of covering many different FMDV serotypes and strains. The producers should respect the strict FMD biosecurity rules worked out by the FAO EUFMD and described in Council Directive 2003/85/EC. Making the experience related to vaccine QA/QC available to all countries will reduce the risk of an FMD outbreak within these countries and consequently will reduce the FMD risk around the world.

Marker vaccines and the impact of their use on diagnosis and prophylactic measures.

Molecular biology and technical advances in DNA recombination have ushered in a new era in vaccinology. This article examines the recent development of specific marker vaccines and examines the impact of their use on the diagnosis and prevention of major infectious diseases. Gene-deleted vaccines, DIVA strategies (differentiating infected from vaccinated animals) and similar methods have been successfully applied in the control and eradication of Aujeszky's disease, infectious bovine rhinotracheitis, classical swine fever, foot and mouth disease and, recently, avian influenza. The efficacy and performance of existing marker vaccines and their companion diagnostic tools (which should be assesed by an independent body) are discussed, as are the ways in which these tools are deployed by competent authorities. The limits and the advantages of the use of marker vaccines are carefully analysed in the light of practical experiences. Although these vaccines can limit the speed and the extent of virus dissemination and thus reduce the number of animals slaughtered, marker vaccines are no substitute for sanitary measures. Early detection and warning systems and the quick implementation of sanitary measures, including stamping out, remain key issues in the control of highly contagious diseases.

Authorisation within the European Union of vaccines against antigenically variable viruses responsible for major epizootic diseases.

Antigenically variable viruses are responsible for some of the most contagious and economically important diseases that affect domestic livestock. The serious consequences of such diseases in terms of economic loss, and human and animal health, were clearly demonstrated by recent epizootics of foot and mouth disease, and outbreaks of avian influenza and bluetongue in the European Union (EU). For such diseases, government authorities need to be able to respond, if appropriate, by making use of vaccines that are suited to the epidemiological situation. The current EU regulatory framework is not well adapted for approval and maintenance of vaccines where the antigens included have to be chosen to reflect the epidemiological need. An extensive revision of the technical requirements for authorisation of veterinary medicinal products within the EU is currently underway. Additionally, a major revision of the regulations that control how such authorisations are kept up-to-date is about to start. This provides an ideal opportunity to introduce into EU legislation the concept of the 'multistrain dossier' whereby a potentially large number of approved strains may be included within a marketing authorisation and the final vaccines may be blended to include strains according to need. In addition, new strains may be added onto the marketing authorisation by means of a rapid regulatory procedure should new antigenic variants actually or potentially threaten the EU.

Validation of a foot-and-mouth disease antibody screening solid-phase competition ELISA (SPCE).

This paper describes the validation of a solid-phase competition enzyme-linked immunosorbent assay (SPCE) for the serological detection of antibody to serotype O foot-and-mouth disease (FMD) in sheep, cattle and pigs. The specificity of the SPCE was calculated from the results of testing known negative sera from sheep, cattle and pigs (n=3030, 1418 and 1495, respectively). The mean percentage inhibition (PI) for known negative sheep, cattle and pig sera were 19.3, 24.1 and 20.8%, respectively. The specificity of the SPCE at a cut-off point (COP) of 60 PI was 99.50% for sheep sera, 99.44% for cattle sera and 100% for pig sera. The analytical sensitivity of the SPCE was examined by testing sera from sheep, cattle and pigs. Based on the testing of serial bleeds from experimentally infected animals, seroconversion at the 60 PI COP occurred between 4 and 9 days post-infection or -exposure, similar to that observed using the virus neutralisation test (VNT) with a COP of 1/45. When applied to 267 sheep and 143 pig samples, that were obtained in Great Britain (GB) during the 2001 FMD UK outbreak, the SPCE identified more positive samples than did the VNT. Estimates of the accuracy, repeatability and reproducibility of the SPCE were verified during the large-scale serosurveillance necessitated by the 2001 outbreak. Results from field and experimental sera showed that when compared against the VNT, the sensitivity of the SPCE was less affected by the choice of virus strain used in the test. Using the O(1) UKG 2001 FMD virus in the VNT with samples representative of the uninfected GB sheep population, the test specificity was 100% at a COP of 1/45.

Persistent infection is a rare sequel following infection of pigs with swine vesicular disease virus.

Nine isolates from pigs persistently infected with a recent Italian isolate of swine vesicular disease (SVD) virus, ITL/9/93, were collected sequentially over 121 days and were characterized antigenically and biochemically. There was an accumulation of amino acid (aa) substitutions in the capsid proteins throughout the carrier state that could be correlated with alterations in antigenicity in virus isolates collected late stage in infection. The aa substitutions detected mainly occurred in VPI and antigenic changes were detected in late isolates both at antigenic site 1, resulting in loss of binding of Mab 4GO7, and at a closely located site which has not yet been named, recognized by Mab C29. In further experiments groups of pigs were exposed to a range of SVD viruses, but no virus was isolated beyond 16 days post infection (dpi) nor viral RNA detected beyond 42 dpi. Attempts to transfer infection to sentinel pigs introduced some time after initial infection of the original pigs were largely unsuccessful. The carrier state was established in only one out of five experimental infections of pigs with SVD virus and can therefore be considered a rare sequel toinfection with SVD virus and is of limited significance in the epidemiology of the disease.

A solid-phase competition ELISA for measuring antibody to foot-and-mouth disease virus.

A solid-phase competition ELISA has been developed to measure antibodies to foot-and-mouth disease (FMD) virus and has been validated using an extensive range of sera from cattle. The assay uses polyclonal antisera and inactivated purified 146S antigens of FMD virus and was compared with the liquid-phase blocking ELISA and the virus neutralisation test on a range of serum sets. When examining test sera at a 1:5 dilution with a cut-off point of 30% inhibition of reaction, the solid-phase competition ELISA was as sensitive as the liquid-phase blocking ELISA for sera from infected or vaccinated animals. The limit of detection of the solid-phase ELISA was similar to that of the liquid-phase assay and both tests had lower limit of detection (i.e. were able to detect lower amounts of antibody) than the virus neutralisation test. The specificity of the solid-phase ELISA was considerably higher than that of the liquid-phase blocking ELISA and almost equivalent to that of the virus neutralisation test. The assay thus retains the sensitivity of the liquid-phase blocking ELISA whilst being easier to use, more robust and specific, and therefore offers an improvement for FMD virus antibody detection.

An integrated model to predict the atmospheric spread of foot-and-mouth disease virus.

The application of a computer model called Rimpuff for simulating the airborne spread of foot-and-mouth disease (FMD) is described. Rimpuff is more sophisticated and accurate than other FMD simulation models previously described. It can be run on a desktop computer and performs analyses very quickly. It can be linked to a geographical information system and so the information generated can be integrated with geographical and demographical data for display in a format that can be easily assimilated and transmitted electronically. The system was validated using historical data from outbreaks of FMD in France and the UK in 1981, and from Denmark and the former German Democratic Republic (GDR) in 1982. A very good fit was obtained between the direction of the plumes of virus simulated by the model and the spread of disease from France to the UK in 1981. Although cattle in the UK were infected during the episode, the concentrations of airborne virus in the plumes simulated by the model were beneath the infectivity threshold for cattle. It was concluded from the analysis that the number of pigs infected in France, and therefore the source concentration of airborne virus, was probably much higher than was recorded at the time of the outbreaks. Analysis of the Denmark/GDR episode pointed to the possibility that the source of virus for the 1982 epidemic in Denmark could have been one or more unreported outbreaks involving pigs in the former GDR.

Validation of the specific isotype assay to detect antibodies against foot-and-mouth disease virus in bovine milk.

The specific isotype assay (SIA) detects IgG1 against foot-and-mouth disease (FMD) virus in bovine milk. A strong correlation was demonstrated between milk antibody titres, and those in serum as measured by the liquid phase blocking ELISA. Thus the SIA would be useful on a herd basis to monitor the milk of vaccinated cattle to determine when re-immunisation is advisable. The SIA titration ELISA was then simplified to a single dilution test and optimised to differentiate the reactions in the milk of FMD-naive cows from those in animals which had been infected with FMD or vaccinated against the disease. For milk from immunised cattle, the pH of the sample was important and borderline positive specimens with a pH of 6.0 or below gave negative results. For milk from naive animals, the optical density (OD) registered in the SIA varied according to the time of year that samples were collected which, in turn, influenced the OD above which milks might be considered positive. Studies showed that the pH of milk could be maintained within the range suitable for the SIA by either storing for up to 1 week at 4 degrees C or by freezing at -20 degrees C for an indefinite period.

Genetic analysis of type O viruses responsible for epidemics of foot-and-mouth disease in North Africa.

The nucleotide sequences of the 3' end of the capsid-coding region were determined for 30 serotype O foot-and-mouth disease (FMD) viruses isolated between 1987 and 1994 from outbreaks in North Africa and the Middle East. These sequences were compared with the previously published sequences of 9 field virus isolates from the Middle East and 5 vaccine virus strains, 3 of which originated from the Middle East (O1/Turkey/Manisa/69, O1/Sharquia/Egypt/72 and O1/Israel/2/85) and 2 from Europe (O1/Lausanne/Switzerland/65 and O2/Brescia/Italy/47). Cluster analysis of these sequences using the unweighted pair group mean average (UPGMA) method showed: (i) that the FMD viruses isolated from North Africa and the Middle East were very different from the classical European vaccine strains; (ii) that all the viruses isolated during the 1989-92 North African epidemic formed a cluster differing by no more than 6% from each other; (iii) a virus isolated in Libya in 1988 was unrelated to the aforementioned epidemic; and (iv) viruses from a second, less extensive epidemic, occurring in 1994, fell into yet another cluster.

Identification of neutralizing epitopes on a European strain of swine vesicular disease virus.

Six neutralizing monoclonal antibodies (MAbs) were used to isolate MAb neutralization-resistant (MAR) mutants from a recent European strain of swine vesicular disease virus (SVDV), ITL/9/93. Sequencing of MAR mutants identified two epitopes located at positions analogous to sites 2A (VP2) and 3B (VP3) on poliovirus (PV) which have been previously identified on a Japanese strain of SVDV. A third epitope near to the C terminus of VP1, not previously recognized on SVDV, was tentatively identified in a region analogous to site 1 of PV. A fourth epitope, located in the C-terminal region of VP3, has never before been recognized as a site of neutralization on picornaviruses. All four epitopes were predicted to be surface-exposed.

The persistence of swine vesicular disease virus infection in pigs.

Two groups of pigs were infected with a recent Italian isolate of swine vesicular disease virus (SVDV). Blood, nasal swabs and faeces were collected for up to 6 months after exposure to infection and animals were killed at regular intervals to obtain tissues post-mortem. These samples were examined for virus by conventional means and for viral RNA (vRNA) by reverse transcription-nested polymerase chain reaction (RT-nPCR). Virus was identified intermittently from both clinically and subclinically infected animals in nasal swabs, faeces and tonsillar tissue by either virus isolation or RT-nPCR up to 63 days post infection (dpi). Between 63 and 119 dpi virus was not detected in the secretions, excretions or tissues of any pigs. Following mixing of the two groups of animals at 119 dpi, SVDV was again identified in faeces for up to 7 days suggesting that the stress of mixing reactivated the excretion of virus in pigs from which the agent could no longer be identified. Minor antigenic changes were identified between the parental virus and isolates recovered late in the course of infection. Altered antigenicity corresponded with deduced amino acid substitutions identified from differences in nucleotide sequence between early and late isolates. This investigation demonstrates that SVDV and vRNA can be present in pigs for considerably longer after exposure to infection than has previously been recognized and provides preliminary evidence for a carrier state in swine vesicular disease.

Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus.

The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins. The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.

Antibody to the nonstructural proteins of foot-and-mouth disease virus in vaccinated animals exposed to infection.

Cattle which have been infected with foot-and-mouth disease (FMD) virus can be differentiated from those that have been vaccinated on the basis of the detection of antibody to one or more of the non-structural (NS) proteins of the virus. Cattle which have been protected by vaccination can become persistently infected with FMD virus (FMDV) without ever showing clinical signs. Vaccinated, protected cattle which are persistently infected cannot be distinguished from animals that merely have been vaccinated on the basis of serological tests for antibody to the structural proteins of FMDV. Sera were collected from groups of cattle for varying periods after exposure to infection under experimental conditions. On the basis of isolation of virus from probang samples collected during the course of the experiments it was possible to classify the cattle according to the following criteria; naive, infected and eliminated the virus (convalescent), infected and persistently infected with FMDV (carriers), vaccinated alone, vaccinated and either convalescent or carrier. Sera were examined for antibody to the NS proteins Lb, 2C, 3A, 3D, and 3ABC by an indirect profiling ELISA using E. coli-expressed fusion proteins as antigens. Considerable variation was observed in the antibody response to NS proteins of both naive and vaccinated animals following infection. The extent of individual variation was so great that convalescent animals could not be differentiated from carrier animals on the basis of their antibody response to any of the NS proteins examined. The majority of vaccinated, protected animals showed an antibody response to NS proteins, particularly 3ABC, following exposure to infection. However, the carrier state was demonstrated in some vaccinated, protected animals in which no antibody response to any of the NS proteins could be detected. The detection of antibody to NS proteins can therefore be used on a group, or herd, basis to detect circulation of virus in a vaccinated population but further investigations in the field are required to determine the sampling level necessary for statistical acceptance. On an individual animal basis, however, freedom from antibody to NS proteins in a vaccinated animal, or an animal of unknown history, does not necessarily imply that the animal is free from infection with FMD virus and, furthermore, the titre of antibody to NS proteins is not a useful predictive measure of whether or not an infected animal has successfully eliminated the virus.