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In order to estimate prevalence, levels and patterns of Listeria monocytogenes contamination, a total of 774 swine carcasses were traced along the Parma ham production chain. Analyses were conducted on isolates originated from the same carcass, collected at different stages during processing, resulting in 0.2% (faeces at intestine removal from carcasses), 3.0% (swabbing of carcasses), 12.5% (fresh hams) and 2.0% prevalence (dry-cured hams). The highest contamination levels of L. monocytogenes were reached in fresh hams after cutting and were followed by a marked decrease during the subsequent processing stages. All the 132 isolates were characterized by serotyping and Pulsed-Field Gel Electrophoresis (PFGE). Transfer of L. monocytogenes between different stages of the processing chain was not reported, whereas processing itself has proved to be an important cause of contamination. The sole isolate of fecal origin belonged to a pulsotype that was uncommon to any of those recovered in carcasses, fresh hams and dry-cured hams, indicating that contaminations from farms does not significantly affect Parma ham production. For the majority of the strains isolated from the same production plants, PFGE profiles were highly similar. In several cases, the same pulsotypes were recurrently detected, over time, in carcasses and fresh ham samples sharing the same processing environment. Aw levels were also measured, showing that drying of the ham surface was able to induce a considerable decrease of the contamination levels, although unable to ultimately remove L. monocytogenes.

Summary Dourine is trypanosomosis that affects equids, it's mainly sexually transmitted. The disease was first eradicated in Italy in the 1940s, but there was then a serious epidemic in the mid-70s. After sporadic reports at the end of the 1990s, in May 2011 it was reported once more. Clinical diagnosis of dourine can be complex, as clinical signs and gross lesions are not always present. Direct laboratory diagnosis is also problematic, given the low number of parasites normally present in infected tissues and the mild, short-lasting parasitaemia. This article describes the epidemiological, clinical and laboratory data enabling confirmation of the suspicion of dourine in Italy in the 2011 epidemic.

Free trade of animals and their products is based on the international or bilateral recognition of the health status of the animal populations being traded. This recognition is based on documentation of their health status by the exporting country, based on the results of continuing surveillance. According to the Terrestrial Animal Health Code of the World Organisation for Animal Health (OIE), this may be based on various methods of surveillance, such as: documenting non-specific surveillance (clinical surveillance, passive notification of suspect cases, etc.); documenting activities that increase the sensitivity of non-specific surveillance (training activities, rewards/sanctions for notification/failure to notify, etc.); documenting all specific surveillance and its results (random surveys, targeted and risk-based surveillance, convenience-testing activities, etc.). Usually, the infection is the subject of the declaration of freedom. While clinical and passive surveillance can provide a high level of confidence that foot and mouth disease (FMD) infection is absent, this is not the case in vaccinated populations. In these populations, specific surveillance becomes much more important than non-specific clinical surveillance. Specific surveillance is severely restricted by the performance of the test(s) employed. The imperfect specificity of any serological test is further complicated when techniques to differentiate infected from vaccinated animals (DIVA) are used, because imperfect purification of the antigen used for vaccination may foster the production of undesired antibodies in the vaccinated animals. The authors discuss various approaches to overcome this problem; their merits and flaws in documenting the absence of infection or virus circulation for animal diseases in general, and for FMD in particular. Particular attention is paid to finding methods that can be applied in a variety of epidemiological conditions and organisational structures, since these vary greatly among OIE Members.

A capture operation to ascertain health status in free-ranging buffaloes from six different areas in the Caprivi Strip in the northeast corner of Namibia was conducted in October 2009. Basic information on the ticks and tick-borne pathogens normally found in wildlife from this area are scarce. The objective of this study was to assess the host status of African buffaloes, Syncerus caffer, for ixodid ticks and two selected tick-borne pathogens in the Caprivi Strip, a key area bordering Angola, Zambia, Botswana, and Zimbabwe. Four different tick species have been identified among the 233 collected specimens, and, of 95 tested buffaloes, 54 (57%) were positive for Theileria parva, whereas only 3 (3%) showed evidence of being infected with Ehrlichia ruminantium.

The bluetongue (BT) epidemic that has prevailed in Europe since 2000 is the first example of continental spread of the BT virus (BTV) in large naive populations of susceptible animals. Based on the results of intensive surveillance and research in countries of the southern Mediterranean that were affected by the infection early on in the epidemic, a new strategy for prevention and control of the disease was developed to limit direct losses and to reduce the consequences due to movement restrictions. The basic innovations that were introduced were the use of mass vaccination of all domestic ruminant species to limit the spread of BTV and the use of intensive active surveillance to limit, as far as possible, the zone where movement restrictions must be applied. The novel strategy that was adopted dramatically reduced the number of clinical outbreaks in southern Europe and the Mediterranean Basin and ensured safer animal trade. In 2006, the first BTV-8 epidemic occurred, this time in north-western Europe. During this epidemic, affected countries adopted a 'wait and see' approach. No vaccination was implemented until 2008 and, in many instances, the movement of animals was authorised within restricted areas, thereby facilitating the spread of infection. The delay in administering vaccination was due to the decision to avoid the use of modified live virus vaccines, although this type of vaccine performed satisfactorily in the previous BT epidemics in southern Europe. Bluetongue has demonstrated that the infectious agents present in southern Africa can easily spread to the Mediterranean Basin, which should be considered a single entity as far as the epidemiology of animal diseases is concerned. Therefore, any effective strategy for the prevention and control of animal disease in Europe must take into account this reality and recognise the need for regional surveillance networks that include all the countries that border the Mediterranean.

Summary To assess the immunogenicity of Zulvac ! 8 Bovis (a commercial inactivated vaccine against bluetongue virus serotype 8 ‐ BTV8) under field conditions, 71 cattle vaccinated according to manufacturer schedule in Verona province (Italy) were tested for the presence of BTV8 neutralizing antibodies at 21, 29, 36, 43, 49, 102 and 201 days post-vaccination (dpv). Another group of 528 BTV8 vaccinated cattle in Mantova province (Italy) was also tested once between 113 and 174 dpv. The vaccine was able to elicit an immune response in 69 (97.2%) and 346 (65.5%) animals of the Verona and Mantova groups, respectively.

Since 2000 severe, long-lasting epidemics of bluetongue virus (BTV) have been described in Italy, caused by BTV serotypes 2, 4, 9 and 16. Vaccination programs have been applied extensively to control the infection, in spite of concerns about the potential dissemination of attenuated vaccine strains of BTV in susceptible animals. Accordingly, rapid and reliable differentiation between vaccine and field strains is paramount in routine diagnosis of BTV to evaluate the extent of this phenomenon. In the present study, we report the development of two real-time RT-PCR assays able to recognise BTV serotypes 2 and 9, respectively, and we evaluated the use of the assays for discrimination between field and vaccine strains. A total of 65 samples collected in Italy from 2000 to 2006 and diagnosed as positive for either BTV-2 or -9 were analysed by the TaqMan assays. Both the assays were found to be highly sensitive and reproducible, ensuring correct serotype characterisation and prediction of the origin of the strains, as confirmed by characterisation using virus neutralisation and sequencing.

The rationale for establishing trade 'regions' and 'zones' is based on principles of epidemiological science and risk analysis that assess and manage animal disease risks so that the safety of trade can be ensured. However, the boundaries of geographical regions and zones may readily be breached through numerous epidemiological pathways. The concept of a 'compartment' extends the application of a 'risk boundary' beyond that of a geographical interface and considers all epidemiological factors that can contribute to the creation of an effective boundary. The fundamental requirement for application of either concept (regions/zones or compartments) is that the population considered for trade is maintained within management or geographical boundaries which allow clear epidemiological differentiation to be made between those animals and surrounding populations of higher risk. Seven factors are presented that an exporting country might use to guide the identification and documentation of a compartment. Additionally, the steps that would be undertaken to implement trade based on the compartmentalisation concept are discussed.

We have measured the content of polychlorinated dibenzo- p -dioxins, and polychlorinated dibenzofurans (together defined as “dioxins”) in 269 samples of food of animal origin collected through the regional veterinary services, covering the national territory. Quantification of the dioxins was accomplished by isotope dilution method, and toxic equivalents (TEQ) were calculated. The average daily food intake was obtained from two main sources: national data collected by the National Institute of Nutrition, and data from an ongoing cohort study on diet and cancer including 40, 000 Italian subjects. The mean value of dioxins measured in food of animal origin was 0.144 ± 0.266 pg-TEQ/g (range: 0.003–1.655 pg-TEQ/g). Fish was the item with the highest content. The estimated intake of dioxins with main food items of animal origin is presented. The major contribution to dioxins intake with food comes from cow milk and fish consumption. These results are in agreement with what observed in studies conducted in other countries, such as Germany, Finland, Japan, Spain, and are below the limits set by the European legislation.

The authors studied the persistence of infection in 46 ewes experimentally infected with Brucella melitensis biovar 3 and monitored through three subsequent reproductive cycles. The entire experimental period lasted for 151 weeks. Infection of ewes and elimination of Brucella in milk, or its presence in vaginal discharges, persisted throughout the duration of the trial, as demonstrated by recurrent elimination of Brucella in milk and vaginal discharges. Brucella melitensis was recovered from the tissues of one ewe killed at the end of the trial. The strain was recovered from vaginal swabs and milk following parturition in the third reproductive cycle from an ewe that had aborted in the first cycle but was not pregnant in the second cycle. From a public health point of view, the periodical recovery of Brucella from the milk during the entire trial period illustrated that brucellosis in sheep remains a continuous occupational risk and a significant public health problem for consumers of fresh milk and milk products. That risk may persist for at least 3 years following the initial infection of the flock. Lamb antibody titres became negative in all lambs within 5 months after birth. This suggested that serological tests on lambs may have no practical diagnostic significance if performed during the first 5 months of life. Nevertheless, the birth of three infected lambs suggested that the phenomenon of latent carrier state may represent another way for B. melitensis to persist in a flock.

Effective implementation and enforcement of legislation is essential to ensure animal welfare. In the World Organisation for Animal Health (OIE) European Region the well-established body of national and European Union laws in existence is growing continuously. The growth is due to various factors, such as new technology in animal farming and experimentation, exploitation of wildlife, new understanding of animal needs, and increasing public awareness and concern. The latter, in particular, determines the need for new animal welfare legislation to regulate and discipline the 'use' of animals for different purposes, such as food production, companionship, work and leisure. This paper intends to provide an overview of the more relevant activities carried out by the Council of Europe and the European Union in the field of animal welfare. The authors identify eLearning as a tool to harmonise the interpretation and the implementation of animal welfare legislation.

Summary In the European Union the serological diagnosis of brucellosis caused by Brucella melitensis is performed using the heterologous antigen of B. abortus S99. The possible higher sensitivity or ability of an early detection of antibodies by a homologous antigen may prove very useful in the final phases of an eradication programme. Results obtained in sheep experimentally infected by B. melitensis biovar 3 were compared using B. abortus S99, B. melitensis M1, M2 and M3 antigens in the Rose Bengal plate test (RBPT), the complement fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA) test. Forty-six sheep from an officially brucellosis-free flock were experimentally infected intraconjunctivally with B. melitensis biovar 3. Prior to infection, all animals were tested first against Brucella antibodies, weekly for 2 months post-infection (PI) and then monthly for a further 7 months. All sera were tested against the antigens listed above using RBPT, CFT and ELISA. Using a Bayesian approach, test sensitivities were estimated and compared. Their ability for the early detection of antibodies was evaluated through a regression model based on a logit response model, using the number of days PI as the independent variable and the logit of the fraction of positive animals as the dependent variable. No significant differences were detected among the various antigens used, either in terms of sensitivity or in terms of antibody kinetics; however, the CFT was significantly less sensitive than the RBPT and ELISA and it also showed a lower rate of increase of percentage positive animals (β-coefficient of regression analysis).

The current outbreaks of bluetongue (BT) disease in sheep in the central parts of the Mediterranean basin are being combated by extensive vaccination to control further spread of the virus and to suppress its long-term maintenance in the field. To be able to monitor the success of this campaign, and to be able to identify new foci of the disease, it is necessary to harness diagnostic methods, both rapid and sensitive, for differentiating reliably field from vaccine strains of bluetongue virus (BTV). A new method is described for their differentiation using fluorescence resonance energy transfer (FRET) probes with real-time PCR. The method is based on the principle that the melting temperature of a DNA duplex gives information about the sequence, and allows even double-base alterations in the amplicon to be identified. The RT-PCR, the generation of melting curves, and fluorescence detection were all performed using the LightCycler system (Roche Diagnostics, Mannheim, Germany).

It is well known that the immune response in sheep against Brucella melitensis is subject to individual variation, depending on diverse factors. It bears asking whether these factors (e.g. clinical disease, active infection, state of previous immunity), when affecting a group, can cause variation in the performance of different diagnostic tests. To clarify some of the circumstances in which this immune response can vary, we examine the immune-response profile of sheep protected against the clinical disease by prior vaccination with strain Rev. 1 in comparison with the profile of unprotected females showing the classical brucellosis symptoms. An experimental infection was provoked at midpregnancy under controlled conditions of both non-vaccinated (n=7) and previously Rev.1-vaccinated ewes (n=5). Their immune response was monitored from 7 to 9 weeks before abortion or normal birth to 30 weeks afterwards. Antibody response was assessed by classical tests (Rose Bengal test, complement fixation test (CFT)) in comparison with other diagnostic tests (indirect ELISA (iELISA), competitive ELISA (cELISA), fluorescence polarization assay (FPA), immunocapture test (ICT)). In addition, the cell-mediated immune response was indirectly evaluated by the in vitro antigen-specific release of gamma-interferon. The antibody levels and antigen-specific gamma-IFN profile of the non-vaccinated ewes having the disease and excreting the pathogen was notably high and differed significantly (P

The authors describe the role of Veterinary Services in dealing with developments in society and the challenges they present. In an increasingly globalised world, in which there are new methods of production, a new and changing relationship between humans and animals and increasingly complex and global problems, the protection of public health and the safety of the environment in which people and animals live require new skills, new knowledge and new technical-scientific responses. Official Veterinary Services are responsible for maintaining hygiene and animal health standards in primary and secondary production and ensuring fair trade. To achieve this, Veterinary Services must ensure that their organisational structures meet internationally recognised quality assurance standards.

Summary In August 2000 bluetongue (BT) disease appeared amongst sheep on the island of Sardinia spreading later to Sicily and to mainland Italy. The majority of areas affected by BT were surveyed for Culicoides imicola, the only proven vector of the disease known to occur in the Mediterranean region. The data from 1456 light-trap collections, made in months with a mean temperature of 12.5°C, were used to test the accuracy of current models predicting the prevalence and abundance of C. imicola across the region. For Italy, the distribution of C. imicola was found to be very irregular and did not fit the modelled predictions. The possible reasons for this are discussed, and suggestions made as to which variables may improve this fit in the development of future risk models. In Italy, past surveys failed to reveal the presence of C. imicola, and so could be construed as evidence of its recent invasion, and thus rampant spread northwards. Although equivocal, historical records indicate that C. imicola was overlooked in the past. Six recommendations are made as to the possible future course of Culicoides research in southern Europe.

Traceability of animals and animal products has become a priority for governments of the developed countries, due to consumer demand for comprehensive and integrated food safety policies. In addition to analysing the differences between traceback and traceability systems, the authors describe some applications of animal traceback systems and the principal characteristics of an animal identification and registration system. The importance of a traceability system for food-borne risk assessment and management, and the most recent approaches towards a comprehensive and integrated animal health and food safety policy are reported.

A telematic system to support decisions and operations in case of animal health emergencies has been designed and implemented in the Abruzzo region of Italy. The system aims to improve decision-making by Veterinary Services in the event of an outbreak of exotic disease. The system has been tested, first by a simulated outbreak of foot and mouth disease, and then during an outbreak of swine vesicular disease. Critical problems were detected and corrected in both cases.

A proposal for the harmonization of quality assurance of Veterinary Services at the international level is made. This proposal is based on the hypothesis of accreditation of Veterinary Services according to the 9000 series of the International Standards Organisation (ISO) standards. An example of a way in which ISO 9000 standards can be used within the context of management of Veterinary Services is given, together with an explanation of the possible role of the Office International des Epizooties in ensuring fairness of evaluations of Veterinary Services at the international level.

The authors propose a contribution to the possible revision of Chapters 1.4.1. and 1.4.2. of the International Animal Health Code (Code) of the Office International des Epizooties (OIE). In particular, data are presented to illustrate some of the inadequacies of both the rationale and the results of the method for risk assessment reported in the Code. The method suggested by the Code for risk assessment is based on the calculation of the 'probability of the occurrence of at least one outbreak' of a given disease following the importation of a given quantity of either live animals or animal products (unrestricted risk estimate). This is usually undertaken when dealing with rare events. For a country such as Italy, this method may not be particularly useful as the frequency of disease outbreaks is what should be estimated, so as to provide decision makers with appropriate and relevant information. Practical use of risk information generated by the use of the OIE risk assessment method for swine vesicular disease (SVD) would have encouraged the Chief Veterinary Officer of Italy to prohibit all imports of swine from the Netherlands and Belgium for at least two years in the early 1990s, with the consequential heavy economic losses for both Italy and the exporting countries. On the contrary, the number of actual outbreaks of the disease due to direct imports of swine from Member States of the European Union (EU), which occurred in Italy in 1992, 1993 and 1994 was very low (two to five outbreaks due to direct imports of swine from the Netherlands and one to two from Belgium). An example of a method for assessing the risks associated with high volumes of trade in commodities is also described. This method is based on the Monte Carlo simulation and provides the information required to evaluate the costs of the strategies compared. The method can be used to predict the number of outbreaks which are likely to occur following importation and enables a comparison to be made of alternative safeguards. This would lead to the selection of the most cost-effective one. The comparison is conducted using risk curves and allows a quantitative evaluation and comparison to be made of various scenarios, varying from an absence of safeguards to combinations of various safeguards.

In 1990 an outbreak of contagious bovine pleuropneumonia (CBPP) occurred in Italy. Subsequent surveillance for CBPP was based on random sampling in bovine herds, serological controls on all animals moved from the herd of origin and controls on slaughtered animals. Official tests employed were the complement fixation test (CFT) and bacteriological isolation and typing. A total of 33,856 serum samples collected from herds in CBPP-free regions were used to define CFT specificity, while samples from 595 animals from infected herds were employed to define the sensitivity. Ninety-nine animals from three infected herds were used to estimate the sensitivity of the isolation technique. Results showed the specificity of CFT (threshold +1:10) to be 98% and sensitivity to be 63.79%. The sensitivity of the test did not change significantly, regardless of whether the lesions were caused by acute or chronic infection. The sensitivity of the isolation technique was 54.1%.

The authors discuss the responsibility of veterinary diagnostic laboratories as suppliers of analytical data for tests on animals and animal products. The guarantee of the quality of analytical data is a basic quality requirement for veterinary certification. It is therefore important for the laboratory to adopt operational quality assurance standards which are recognised internationally. The management and quality assurance criteria contained in the International Standardisation Organisation/International Electrotechnical Commission Guide 25, or in directives or guidelines established by international organisations such as the Office International des Epizooties and the Codex Alimentarius of the Food and Agriculture Organisation, are reviewed. These documents provide procedures for adopting the principles of quality assurance in order to acquire the recognition of competence to execute the laboratory tests required for national and international veterinary certification.

This report describes adverse events to vaccination in eight children in southern Italy. These children were seen in the Emergency Room of a hospital of the Local Health Unit SA2 (Salerno, Italy) after administration of hexavalent vaccine (Hexavac, Aventis-Pasteur). On analysing available documentation on the adverse reactions (label instructions, Medline), the Authors concluded the cases reported were to be expected. However, the label instructions did not give sufficient information on the type and frequency of adverse reactions. If the label instructions are to furnish complete, operative information for the vaccination units, the Italian instructions for the Hexavac vaccine can be considered inadequate.

Free trade of animals and their products is based on the international or bilateral recognition of the health status of the animal populations being traded. This recognition is based on documentation of their health status by the exporting country, based on the results of continuing surveillance. According to the Terrestrial Animal Health Code of the World Organisation for Animal Health (OIE), this may be based on various methods of surveillance, such as: documenting non-specific surveillance (clinical surveillance, passive notification of suspect cases, etc.); documenting activities that increase the sensitivity of non-specific surveillance (training activities, rewards/sanctions for notification/failure to notify, etc.); documenting all specific surveillance and its results (random surveys, targeted and risk-based surveillance, convenience-testing activities, etc.). Usually, the infection is the subject of the declaration of freedom. While clinical and passive surveillance can provide a high level of confidence that foot and mouth disease (FMD) infection is absent, this is not the case in vaccinated populations. In these populations, specific surveillance becomes much more important than non-specific clinical surveillance. Specific surveillance is severely restricted by the performance of the test(s) employed. The imperfect specificity of any serological test is further complicated when techniques to differentiate infected from vaccinated animals (DIVA) are used, because imperfect purification of the antigen used for vaccination may foster the production of undesired antibodies in the vaccinated animals. The authors discuss various approaches to overcome this problem; their merits and flaws in documenting the absence of infection or virus circulation for animal diseases in general, and for FMD in particular. Particular attention is paid to finding methods that can be applied in a variety of epidemiological conditions and organisational structures, since these vary greatly among OIE Members.

The authors describe the clinical profile and epidemiology of contagious bovine pleuropneumonia in Europe. This disease, once considered to have been eradicated several years ago, has now become endemic in southern countries of Europe. The status of contagious bovine pleuropneumonia in Portugal and Italy, and the evolution of the disease during the last ten years, are analysed in detail, in addition to the measures undertaken for control and eradication. The authors also refer to hosts and possible reservoirs of the infection.

The bluetongue (BT) epidemic that has prevailed in Europe since 2000 is the first example of continental spread of the BT virus (BTV) in large naive populations of susceptible animals. Based on the results of intensive surveillance and research in countries of the southern Mediterranean that were affected by the infection early on in the epidemic, a new strategy for prevention and control of the disease was developed to limit direct losses and to reduce the consequences due to movement restrictions. The basic innovations that were introduced were the use of mass vaccination of all domestic ruminant species to limit the spread of BTV and the use of intensive active surveillance to limit, as far as possible, the zone where movement restrictions must be applied. The novel strategy that was adopted dramatically reduced the number of clinical outbreaks in southern Europe and the Mediterranean Basin and ensured safer animal trade. In 2006, the first BTV-8 epidemic occurred, this time in north-western Europe. During this epidemic, affected countries adopted a 'wait and see' approach. No vaccination was implemented until 2008 and, in many instances, the movement of animals was authorised within restricted areas, thereby facilitating the spread of infection. The delay in administering vaccination was due to the decision to avoid the use of modified live virus vaccines, although this type of vaccine performed satisfactorily in the previous BT epidemics in southern Europe. Bluetongue has demonstrated that the infectious agents present in southern Africa can easily spread to the Mediterranean Basin, which should be considered a single entity as far as the epidemiology of animal diseases is concerned. Therefore, any effective strategy for the prevention and control of animal disease in Europe must take into account this reality and recognise the need for regional surveillance networks that include all the countries that border the Mediterranean.

Summary The authors report a case of casual isolation of virulent Newcastle disease virus (NDV) from embryonated eggs. The virus was isolated from uninfected chicken embryo liver and fibroblast cultures prepared from commercial embryonated fowls' eggs. A serological and virological investigation carried out on the breeders which had laid those eggs showed high titres against NDV, and virus isolation from cloacal swabs. The virus was also isolated from the progeny of the same breeders which showed no clinical signs of ND, following episodes of mortality. Vertical transmission of the virus is discussed.

To assess the immunogenicity of Zulvac 8 Bovis (a commercial inactivated vaccine against bluetongue virus serotype 8 - BTV8) under field conditions, 71 cattle vaccinated according to manufacturer schedule in Verona province (Italy) were tested for the presence of BTV8 neutralizing antibodies at 21, 29, 36, 43, 49, 102 and 201 days post-vaccination (dpv). Another group of 528 BTV8 vaccinated cattle in Mantova province (Italy) was also tested once between 113 and 174 dpv. The vaccine was able to elicit an immune response in 69 (97.2%) and 346 (65.5%) animals of the Verona and Mantova groups, respectively.

In the European Union, RB51 vaccine can be used only under strictly controlled conditions for the immunisation of cattle at risk of infection with Brucella abortus. A test is therefore necessary to distinguish vaccinated from unvaccinated animals. The complement fixation test with RB51 antigen (RB51-CFT), dot-blot and gamma-interferon used to identify vaccinated animals have been described, but sensitivity of the tests has been poor and positivity transient after calfhood vaccination. To avail of a rapid and accurate diagnostic tool, the authors produced, controlled and evaluated an experimental brucellin prepared from strain RB51 (RB51 brucellin). The potency of this brucellin was evaluated in guinea-pigs sensitised with RB51 and compared with a commercially available brucellin. Both allergens produced similar biological activity in guinea-pigs. The RB51 brucellin skin test was performed in 10 cattle 414 days after calfhood vaccination with RB51 when they were negative to the RB51-CFT. The skin test revealed 60% sensitivity (with a confidence interval of 95%, CI 30.8%-83.3%) and 100% specificity (CI 60.7%-100%). These findings limit the use of the skin test only for screening to detect RB51 vaccinated herds, not individual animals. Nevertheless, following intradermal inoculation of RB51 brucellin, a transient antibody increase to the RB51-CFT was observed, from day 9 to day 20 post inoculation with RB51 brucellin. This transient antibody increase, when evaluated in parallel with the RB51 brucellin skin test results, enables detection of individual vaccinated animals (sensitivity 100%; CI 76.2%-100%).

An indirect ELISA was evaluated for the detection of Brucella antibodies in milk (m-ELISA) from sheep experimentally infected with B. melitensis biovar 3. At the end of the second reproductive cycle (13 months post infection), the milk of 22 lactating sheep was tested using the m-ELISA. Sera from the same sheep were also tested for Brucella antibodies using the Rose Bengal test (RBT) and the complement fixation test (CFT). The first serum sampling after parturition showed 100% sensitivity in both the RBT and the CFT (confidence interval [CI] 94-100%), but in subsequent samplings the sensitivity of the RBT decreased to 73% (CI 55-85%). Similarly, the sensitivity of the CFT decreased two months after the first sampling, when respective sensitivities of 95% (CI 81-98%) and 81% (CI 61-93%) were recorded for the final two samplings. The sensitivity of the m-ELISA decreased initially (68% on the third sampling, CI 50-81%), but then increased to 95% (CI 81-98%) for the final sampling. When disease prevalence in a flock is below 5%, the estimated probability of not detecting an infected flock through m-ELISA bulk milk testing is over 25%. Under field conditions in Italy (average sheep flock size of 70), the probability that the infection will not be detected is over 25% when four (or less) infected milking sheep are present in the flock. The results show that the m-ELISA is not a reliable screening test for bulk milk samples when the prevalence of brucellosis in a sheep flock is low.

An inactivated vaccine was produced from an Italian field isolate of bluetongue virus serotype 2 (BTV-2) with a titre of 10(7.8)TCID50/ml. The virus was purified through a molecular cut cassette membrane, inactivated with beta-propriolactone and emulsified with ISA 206 (Seppic) adjuvant. The vaccine was then tested for sterility, toxicity and safety in laboratory and target animals according to European Pharmacopoeia standards. Immunogenicity was assessed by inoculating subcutaneously 10 sheep and 10 goats each with 2 ml of the vaccine and 10 bovines each with 5 ml of the vaccine. A booster dose was inoculated after 14 days and no side-effects were reported following vaccination. Fourteen days after the booster dose, all vaccinated animals developed virus neutralising (VN) bluetongue (BT) antibody titres that on day 60 post vaccination ranged between 1/20 and 1/1 280. After one year, goats still had high VN antibody titres. Sheep were challenged 138 days after vaccination by subcutaneously inoculating 1 ml of 10(5.6)TCID50/ml of an Italian field isolate of BTV serotype 2; four unvaccinated animals were also inoculated and used as controls. Starting from day 6 post challenge, control animals developed a fever, with temperature ranging from 39.9 degrees C to 40.6 degrees C and lasting 48 h on average. BTV-2 was also isolated from the blood of control animals between days 4 and 20 post challenge. Conversely, neither fever nor viraemia were detected in the vaccinated animals that were challenged. A new trial with a larger number of animals, including all target species, has been planned and is in progress.

As a consequence of the recent outbreaks of bluetongue (BT) disease amongst sheep in the Mediterranean Basin, and following the subsequent vaccination campaign to control further spread of the disease and its long-term maintenance, it has become most important to develop rapid and sensitive methods that can reliably differentiate between field and vaccine strains of the causative virus. The authors describe a new method to differentiate bluetongue virus serotype 2 (BTV-2) field and vaccine strains, using the VP2 gene sequence differences between the South African vaccine and the Italian field wild-type strains. The method is based on the principle that the melting temperature of a DNA duplex gives information on the sequence, which enables the identification of even single-base alterations in the amplicon. The real-time polymerase chain reaction the generation of melting curves and fluorescence detection were all performed using the light cycler system (Roche). Primers and probes were designed using VP2 gene sequences. After RT-PCR, the melting curves analysis, derived by the fluorescence resonance energy transfer (FRET) real-time PCR, was performed using the light cycler data analysis program (Roche). To assess the diagnostic value of the method, a BTV-2 vaccine strain (Onderstepoort Biological Products, South Africa) was first compared against a field strain of BTV-2 (isolated during an outbreak in 2000 in Sardinia). The ability of the method to reliably identify all the BTV-2 strains was tested using an array of eleven BTV-2 field strains isolated during outbreaks in various Italian regions between 2000 and 2002 and other serotypes (BTV-1, BTV-4, BTV-9 and BTV-16) that had been isolated during recent outbreaks of BT in the Mediterranean Basin. The method was clearly able to differentiate BTV-2 strains of vaccine virus from all wild-type strains of the same serotype tested. The resultant melting curves distinctly reveal the two strains to have differing peak values of 47.8 degrees C-/+0.6 degrees C and 60.5 degrees C-/+0.6 degrees C, respectively.

The antibody response following bluetongue (BT) vaccination under both field and experimental conditions, and the duration of colostral antibodies in calves born from vaccinated dams, were evaluated. To this end, 1 005 animals of various breeds and ages were selected at random from 10 herds in the Sardinian province of Oristano. During the first year of the vaccination campaign, the animals selected were vaccinated against BT virus (BTV) serotype 2 between July and August 2002. Blood samples were taken from all animals monthly for three months after vaccination and tested for the presence of BT antibodies using the competitive enzyme-linked immunosorbent assay (c-ELISA) and the virus neutralisation (VN) test. Serological results from field vaccinated animals were compared with those obtained following the vaccination of five animals under experimental conditions. Out of 1,005 animals, 994 (98.1%) developed BT antibody following vaccination whereas antibody was detected in all cows vaccinated under experimental conditions. Both groups showed the highest median titres of 1:160 after two months. To assess the duration of colostral antibodies in calves born from vaccinated dams, the sera of 47 calves were screened using the c-ELISA and VN test. Calves were divided into three age groups: Group A included 22 calves aged 1 to 25 days, Group B 13 calves aged 26 to 39 days and Group C 12 calves aged 40 to 60 days. Antibody was detected in calves in Groups A and B (68.2% and 46.1%, respectively) whereas the calves in Group C were serologically negative.

A challenge study was conducted to determine the efficacy of vaccination against bluetongue (BT) virus (BTV) serotype 2 in protecting cattle against infection. A group of 30 cows, vaccinated seven months previously with monovalent BTV-2 modified-live vaccine produced by Onderstepoort Biological Products in South Africa, were challenged subcutaneously with 2x 10(5.8)TCID50/ml of BTV-2 field isolate. All cattle originated from the same population in the Sardinian province of Oristano. Eight unvaccinated calves from a BTV-free herd also participated in this study; four were inoculated with BTV-2 and used as positive controls whilst the remaining four were used as negative controls to confirm that no BTV was circulating locally. Blood samples were taken from all animals three times a week for two months. Serum samples were tested for antibody against BTV using the competitive enzyme-linked immunosorbent assay (c-ELISA) and the virus neutralisation (VN) test. Virus isolation was attempted on the blood samples by intravenous egg inoculation followed by two blind passages in Vero cells. Virus titres following challenge were determined also. Of the 30 cows vaccinated, 29 were positive in the c-ELISA and demonstrated neutralising antibodies. At the time of challenge, 11 cows had no virus neutralising antibody while the remainder had low titres ranging from 1:10 (11 cows) to 1:20 (6 cows); two cows showed titres of 1:40 and 1:80, respectively. None of the cows showed signs of disease after challenge and no BTV was isolated from the blood of the 29 cows that had developed antibodies after vaccination. Commencing on day 9 post challenge, BTV-2 was isolated from the blood of the single cow that had not seroconverted following vaccination and from the blood of the unvaccinated controls. Viraemia lasted until day 21 post challenge. Neither BTV nor antibody was detected in the blood samples taken from the negative control group. These observations indicate that the monovalent BTV-2 modified-live vaccine protects most animals when challenged with field virus seven months post vaccination.

Since the first appearance of bluetongue (BT) in Italy in late August 2000, the ecology of vectors and the environmental conditions affecting their distribution and survival proved to be the most difficult factors to monitor, and represented a serious challenge to the effectiveness of the National Contingency Plan promulgated in 1991. Moreover, the Italian Ministry of Health considered the national management plan of BT inadequate to prevent further spread of the disease. The authors describe the implementation of a new BT contingency plan, integrating an operations manual and an ad hoc information system, which operated also as a decision-support system both at local and central levels. The plan describes the national capacity for dealing with BT outbreaks, the composition and duties of the National Disease Control Centre and Local Disease Control Centres, the chain of command and the strategies adopted.

Transhumance, or seasonal grazing, in central Italy is a husbandry practice that is over two thousand years old. It involves the seasonal movement of sheep, goats and cattle from the southern lowlands of mainly the Puglia and Lazio regions, to summer pastures in the mountains of Abruzzo and Molise. Bluetongue (BT) made its appearance in Italy in 2000. In the early summer of 2001, disease was present in three regions: Sardinia, Sicily and Calabria. Neither an effective surveillance system nor a vaccination campaign had been implemented. Movement of ruminants to the disease-free regions of Abruzzo and Molise was therefore banned. The Italian Veterinary Services had to meet the challenge of the movement of ruminants from surveillance to disease-free zones, given the impossibility of stopping transhumance. The General Directorate of Veterinary Public Health, Food and Nutrition of the Ministry of Health developed a plan for both the Puglia and Abruzzo regions based on serological, virological and entomological surveillance. The plan was implemented between May and June 2001 when 7,000 animals moved from the Puglia surveillance zone to the infection-free summer pastures. In the early summer of 2002, eight regions were infected (Sardinia, Sicily, Calabria, Basilicata, Puglia, Campania, Lazio and Tuscany). Simultaneously, a nationwide surveillance system and a vaccination campaign, were implemented in infected regions. In the provinces where vaccination was compulsory, deviation from the animal movement ban was allowed if at least 80% of susceptible stock had been vaccinated. However, this objective was not achieved in the provinces of Rome and Viterbo (Lazio) where a large transhumant population was present and where sporadic virus circulation had been detected. A specific control plan to allow transhumance from Lazio to Abruzzo, Marche and Umbria was designed and implemented to increase the number of animals that could be moved. Between May and June 2002, authorisation was granted to move 28,000 head, whereas prohibition of movement was ordered for 12,000 sheep (belonging to 21 flocks). Regional authorities financed feeding, watering and housing for these animals. Transhumance did not spread infection to disease-free areas either in 2001 or in 2002.

The eastern focus of the current outbreak of bluetongue (BT) in the Mediterranean Basin commenced in late 1998, infecting Turkey and some of the eastern islands of Greece. In the summer of 1999 it moved to continental Greece and for the first time to Bulgaria. By the late summer of 2000, BT spread progressively through Greece and to the Balkan states. The BT virus (BTV) serotypes involved were BTV-4, BTV-9 and BTV-16. The west-central focus of the outbreak, involving BTV-2, appeared in Tunisia in December 1999 and the following summer also in Algeria. In August 2000, BTV-2 was reported for the first time in Italy (in Sardinia) and soon thereafter in France (Corsica) and in Spain (the Balearic Isles). In the autumn of 2000, a second serotype (BTV-9) emerged in southern peninsular Italy. Eventually this incursion of virus into the central Mediterranean region resulted in the largest epidemic of BT ever to affect Europe. Some features of this epidemic differ significantly from those observed previously, namely: a) its deep penetration northwards (reaching 44 degrees N both in Italy and in the Balkans) b) its persistence across four seasons in various zones of Italy and the Balkans, implying that BT could become endemic over a wide geographic area c) its successful invasion of areas separated from previously infected ones by fairly large distances (Sardinia, Sicily, Calabria, and the Balearic islands). The pattern of the spread of BT across Italy, before the introduction of vaccination, is described. The possible role of climate, soil and insect vectors on the incidence of the disease, and the overwintering of the virus, are discussed. Some hypotheses on the possible origins and modes of introduction of BTV into Italy are postulated.

Since August 2000, Italy has lost almost 500,000 sheep to the largest incursion of bluetongue (BT) yet to affect Europe. The national BT surveillance system includes serological and entomological programmes. The main objective of the serological programme is the early detection of BT virus (BTV) circulation by periodical testing of more than 30,000 sentinel cattle distributed across Italy. The sentinel network has been in force since October 2001 and since its inception has provided accurate and timely detection of viral circulation. However, the repeated testing of such a large number of animals required significant effort from the Veterinary Services and was costly for farmers. Consequently, a Montecarlo simulation model was developed to simulate different sentinel system scenarios and the results from each were compared. The model was validated using data derived from the serological surveillance activities in Sardinia from October 2001 to December 2002.

In summer 2000, bluetongue (BT) infection was reported in Italy and caused a widespread epidemic involving a total of ten southern and central regions and is still in progress after three years. From the date of the first case (18 August 2000) to 14 May 2001, when the lowpoint in the first epidemic curve was reached, a total of 310,234 animals in 6,869 flocks of three regions had been involved. From 15 May 2001 to 14 April 2002, when a second epidemic wave swept through central and southern Italy, a total of 323,635 animals in 6,807 flocks in seven regions were involved. During 2000 and 2001 virtually no susceptible ruminants were vaccinated. On 11 May 2001, the Italian Ministry of Health ordered the vaccination of all susceptible domestic ruminant species (i.e. sheep, goats, cattle and water buffalo) in the infected and surrounding areas. The vaccination strategy stemmed from a risk assessment that demonstrated the possibility of such a strategy preventing most of the direct economic losses and decreasing the level of virus circulation. Vaccination of the target populations commenced in January 2002. In July 2002, when the new epidemic peak was reached, the percentage of vaccinated populations varied between the regions with direct consequences on the spread of BT. The relationship between vaccination coverage of the target populations and animal losses due to disease and virus circulation, and as detected by the sentinel surveillance system, was analysed. The effectiveness of the vaccination campaign in limiting virus circulation and consequently indirect losses due to animal movement restrictions was analysed and evaluated. At the end of 2002, a second risk assessment led to the authorization of the movement of vaccinated animals from infected areas (where at least 80% of the susceptible population was vaccinated) directly to slaughter in unvaccinated areas free from infection. This risk assessment also generated new criteria to define zones where animal movement restrictions should be applied. Following the second vaccination campaign (January to May 2003), a third risk assessment was performed and the results from vaccination trials performed in controlled and in field conditions studied. These studies indicated that procedures to move vaccinated breeding animals from zones where infection exists to unvaccinated infection free zones could be contemplated.

The authors provide details of the bluetongue surveillance and the Internet-based information systems that were implemented in Italy. The systems were structured with the aim of gathering and spreading information and data to support decision-making, management of control activities and provide an early warning system. Information and data generated by the surveillance system enabled the detailed analysis of bluetongue epidemiology, vector distribution and vector population dynamics. This information and data also allowed the analysis of risk factors associated with vector spread and animal movements, which resulted in and increased the flexibility and the efficiency of the enforcement of control measures.

Summary In the recent years, USUTU virus (USUV), a flavivirus of the Japanese encephalitis virus complex, has been reported in Central Europe. As part of a systematic surveillance programme to monitor possible entrance and/or circulation of vector-borne viruses, since 2001, sentinel-chicken flocks were placed throughout the Italian territory nearby areas considered at risk of virus introduction. They have been periodically checked for the presence of antibodies against flaviviruses by indirect ELISA, plaque reduction neutralization test for USUTU, West Nile and tick-borne encephalitis viruses. In July 2007, a sentinel chicken in a flock of 20 animals located within the Ravenna province seroconverted to USUV reaching neutralizing titres up to 1 : 5120. A second chicken seroconverted to the same virus 2 months later. Although no virus was rescued from these animals and from wild or farm birds sampled in the area, these results still provided evidence of the circulation of USUV in north-eastern Italy.

The efficacy of a bivalent inactivated vaccine against bluetongue virus (BTV) serotypes 2 (BTV-2) and 4 (BTV-4) was evaluated in cattle by general and local examination, serological follow-up, and challenge. Thirty-two 4-month-old calves were randomly allocated into 2 groups of 16 animals each. One group was vaccinated subcutaneously (s/c) with two injections of bivalent inactivated vaccine at a 28-day interval, and the second group was left unvaccinated and used as control. Sixty-five days after first vaccination, 8 vaccinated and 8 unvaccinated calves were s/c challenged with 1 mL of 6.2 Log 10 TCID 50 /mL of an Italian field isolate of BTV serotype 2, while the remaining 8 vaccinated and 8 unvaccinated animals were challenged by 1 mL of 6.2 Log 10 TCID 50 /mL of an Italian field isolate of BTV serotype 4. Three additional calves were included in the study and used as sentinels to confirm that no BTV was circulating locally. At the time of the challenge, only one vaccinated animal did not have neutralizing antibodies against BTV-4, while the remaining 15 showed titres of at least 1:10 for either BTV-2 or BTV-4. However, the BTV-2 component of the inactivated vaccine elicited a stronger immune response in terms of both the number of virus neutralization (VN) positive animals and antibody titres. After challenge, no animal showed signs of disease. Similarly, none of the vaccinated animals developed detectable viraemia while bluetongue virus serotype 2 and 4 titres were detected in the circulating blood of all unvaccinated animals, commencing on day 3 post-challenge and lasting 16 days. It is concluded that administration of the bivalent BTV-2 and BTV-4 inactivated vaccine resulted in a complete prevention of detectable viraemia in all calves when challenged with high doses of BTV-2 or BTV-4.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides var. mycoides small colony (MmmSC), is one of the most important diseases of cattle in Africa. The role of innate or acquired cell mediated and humoral immunity in conferring protection against MmmSC infection has not yet been elucidated. On the other hand, the pathological lesions caused by the aetiological agent have been considered indicative of an immunopathological process. In this study ten naïve cattle were exposed to in-contact infection with animals infected by intubation with a strain of MmmSC. Clinical signs, antibody response, IFNgamma release and pathological changes at necropsy were analysed and compared with the events following in-contact infection of an equal number of animals kept under daily treatment with cyclosporine for the entire observation period of 84 days. Cyclosporine is a suppressor of the immune response related to the T-cell system. Under the conditions of the experiment, cyclosporine appeared to condition the pathogenesis of CBPP by delaying the events that follow infection, bringing further support to the possibility that the immune response may have an impact on the disease outcome.

The rationale for establishing trade 'regions' and 'zones' is based on principles of epidemiological science and risk analysis that assess and manage animal disease risks so that the safety of trade can be ensured. However, the boundaries of geographical regions and zones may readily be breached through numerous epidemiological pathways. The concept of a 'compartment' extends the application of a 'risk boundary' beyond that of a geographical interface and considers all epidemiological factors that can contribute to the creation of an effective boundary. The fundamental requirement for application of either concept (regions/zones or compartments) is that the population considered for trade is maintained within management or geographical boundaries which allow clear epidemiological differentiation to be made between those animals and surrounding populations of higher risk. Seven factors are presented that an exporting country might use to guide the identification and documentation of a compartment. Additionally, the steps that would be undertaken to implement trade based on the compartmentalisation concept are discussed.

The Authors describe an epidemiological survey performed after a cluster of cases of foodborne infection involving several participants at a wedding reception. The aim was to identify the food, the responsible pathogen and any shortcomings in the coordination between the various services and the territorial operating units involved in the outbreak investigation. The investigation involved 149 participants; fifty seven persons (38.3%) had a foodborne illness. The only food item that remained associated with illness after multiple regression analysis was the ricotta cheese (RR 3.58, I.C. 1.,72-7.48 ). The finding of B. cereus in samples of ricotta cheese collected at the dairy food supplier indicate its responsibility for the outbreak but diagnostic certainty could not be achieved as no leftovers were available. Thus, shortcomings in this epidemiological investigation are constituted by: delayed notification, which prevented the acquisition of food samples, lack of a reference laboratory and lack of a specific diagnostic protocols, which prevented the microbiological research for the rapid identification of new pathogens incriminated in foodborne diseases. Finally, environmental and sanitary inspections showed deficiencies in the hygienic measures of food storage, particularly regarding refrigeration and in the HACCP plan that was not properly implemented.

Effective implementation and enforcement of legislation is essential to ensure animal welfare. In the World Organisation for Animal Health (OIE) European Region the well-established body of national and European Union laws in existence is growing continuously. The growth is due to various factors, such as new technology in animal farming and experimentation, exploitation of wildlife, new understanding of animal needs, and increasing public awareness and concern. The latter, in particular, determines the need for new animal welfare legislation to regulate and discipline the 'use' of animals for different purposes, such as food production, companionship, work and leisure. This paper intends to provide an overview of the more relevant activities carried out by the Council of Europe and the European Union in the field of animal welfare. The authors identify eLearning as a tool to harmonise the interpretation and the implementation of animal welfare legislation.