The persistence of Brucella melitensis in experimentally infected ewes through three reproductive cycles.

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.

The use of homologous antigen in the serological diagnosis of brucellosis caused by Brucella melitensis.

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 (beta-coefficient of regression analysis).

Use of the complement fixation and brucellin skin tests to identify cattle vaccinated with Brucella abortus strain RB51.

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%).

Vaccination in the control strategy of bluetongue in Italy.

The incursion of bluetongue (BT) in Italy, in August 2000, caused heavy economic losses, partly due to the direct effect of the disease on the animals, but mostly due to indirect losses due to ruminant movement restrictions conducive to heavy losses to the cattle and sheep industry. To limit losses due to both disease and virus circulation, which was the cause of movement restrictions, the Italian Ministry of Health in May 2001 ordered the vaccination of animals of all domestic ruminant species in infected and "at risk" areas. The vaccination strategy derived from a risk assessment that suggested that the vaccination of all domestic ruminants could reduce both direct losses and virus circulation significantly. The different levels of vaccination coverage, achieved in the various regions of Italy, had clear consequences on the spread of both disease and infection. In regions where more than 80% of the target populations were vaccinated properly, the disease disappeared almost completely and virus circulation was significantly reduced, as documented by the serological surveillance system, after a single vaccination cycle. This led to a significant decrease in the areas subject to movement restrictions. Data generated by both field and controlled experiments contributed to modify the EU approach to BT and to some of the conclusions of the Third OIE International Symposium on Bluetongue that will probably lead to a modification of the Office International des Epizooties (OIE) standard.

Bluetongue virus surveillance in a newly infected area.

The occurrence of bluetongue virus (BTV) in areas in which intensive animal production is practised and where there is extensive movement of animals may have a substantial impact on both animal trade and husbandry. This situation occurred in Italy after the detection of bluetongue (BT) in August 2000. In such situations, surveillance can be used to delineate with precision those areas in which the virus is circulating and, consequently, to enforce the appropriate animal movement restrictions. Furthermore, surveillance can provide the data required to assess the risk associated with animal movement and trade. A structured surveillance system for the detection of BTV has been in place in Italy since August 2001. The system is based on the periodical testing of unvaccinated sentinel cattle that are uniformly scattered throughout Italy in a grid of 400 km(2) cells. The initial number of sentinel sites and sentinel animals, together with the width of the restricted area generated by the finding of a single seroconversion in a sentinel animal, were based on conservative criteria. Animal movement was restricted in a 20 km radius buffer zone around any positive serological result. This buffer area extends about 1,257 km(2), equivalent to the area of three grid cells. After the commencement of the BT vaccination campaign in Italy, the sentinel surveillance system was the only way in which the effectiveness of vaccination and the incidence of infection in the non-immunised strata of ruminant animals could be estimated. Data collected over two years was used to assess the risks posed by the adoption of less conservative criteria for the delineation of infected areas and by the progressive relaxation of movement restrictions of vaccinated animals. In regard to the delineation of restricted areas, a new approach was tested and validated in the field, based on a Bayesian analysis of the positive and negative results obtained by the testing of sentinel animals from defined regions. For the risks related to animal movement, the surveillance data was used in risk assessment analyses to address the movement of slaughter and breeding animals from vaccinated/infected and surrounding areas to free areas. These risk assessments led to an amendment of the relevant European Union legislation. Finally, a Montecarlo simulation model was developed to simulate different sentinel system scenarios and to decrease the total number of sentinel animals and sites required by the surveillance system. The sentinel surveillance system was complemented by an entomological surveillance system based on the use of a number of permanent blacklight traps run weekly year-round and a number of mobile blacklight traps moved through the grid cells during the summer and autumn of each year. The aim of entomological surveillance was to define the maximum distribution of vectors and their seasonal population dynamics. Furthermore, the permanent trap system provides an early warning of the start of new epidemics. The data from the entomological surveillance system were also analysed to generate probability maps of the presence of the principal BTV vector (Culicoides imicola) and to define the geographical risk of BT on a nationwide basis, and to predict the geographical distribution and the short-term spread of C. imicola in Sardinia, using spatio-temporal data. The detection, since 2001, of BT outbreaks in the absence of C. imicola and the recent identification of BTV in midges of the Obsoletus Complex also stimulated investigations on other vector Culicoides, including C. obsoletus and C. pulicaris.

Bluetongue in Italy: Part I.

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.

Bluetongue in Italy: Part II.

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.

Surveillance system of bluetongue in Italy.

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.

Bluetongue vaccination in Europe: the Italian experience.

The incursion of bluetongue (BT) into Italy in August 2000 caused heavy economic losses, partly due to the disease itself, but mostly because of disruption caused to the national animal trade structure. To limit direct losses and the circulation of BT virus (BTV), the Italian Ministry of Health ordered, on 11 May 2001, the vaccination of all susceptible domestic ruminant species (i.e. sheep, goats, cattle and water buffalo) in both infected and surrounding areas. The vaccination strategy was based on a risk assessment that suggested it would prevent direct economic losses and significantly reduce virus circulation. Vaccination of the target animal populations commenced in January 2002, prior to the epidemic peak of BT that began in July 2002. The proportion of vaccinated animals differed between the various regions and the varying levels of vaccination of these populations had clear consequences on the occurrence of clinical disease and the spread of BTV infection. In those regions where more than 80% of the target population were properly vaccinated, the disease disappeared almost completely and virus circulation was reduced significantly. The importance of this reduced circulation of BTV (i.e. infection did not spread from affected areas) was immediately obvious in areas affected by the less virulent BTV serotype 9 where, despite the virtual absence of clinical disease, trade of animals to other areas was prohibited. The areas affected by the highly virulent BTV-2 also benefited from vaccination because it eliminated clinical disease while animal movements were prohibited. The main consequence of the reduction of virus circulation after vaccination, as documented by serological surveillance, was a significantly reduced expansion of the areas that were subjected to animal movement restrictions. Subsequently, analysis of surveillance data, coupled with specific risk assessments, led to a progressive relaxation of movement restrictions even in areas where the infection was still present but where most of the population had been adequately vaccinated. The effectiveness of the strategy used in Italy (i.e. vaccination of all domestic ruminants) was reinforced by extensive experimental and field studies. The aim of these studies was to: a) evaluate levels of individual and herd immunity and resistance to challenge conferred by vaccination, and b) quantify the frequency and severity of the adverse effects of vaccination on domestic ruminants. Ongoing research has focused on the ability of vaccination to suppress or reduce viraemia in ruminants following natural challenge by a virulent BTV strain. These studies address the issue of safety of the trade and movement of vaccinated animals that originate from areas in which BTV continues to circulate and could justify the reversal in current policy that restricts the international trade of animals vaccinated against BT.

Health management of large transhumant animal populations and risk of bluetongue spread to disease-free areas.

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.

Comparison of serological and milk tests for bovine brucellosis using a Monte Carlo simulation model.

European Union (EU) Directive 97/12/EC allows the trade of cattle within the EU of animals originating from an 'officially brucellosis-free herd'. To qualify for this status, a number of different programmes must be implemented. Each EU Member Country is free to decide which procedure to use to qualify herds. The authors conducted a study to compare the merits and costs of testing programmes given in the Directive and of some alternative testing strategies. The effectiveness of testing programmes was evaluated by a Monte Carlo simulation model. Programmes listed in the Directive do not appear to have identical sensitivity and specificity. Simulations of the programmes showed that milk testing may be more effective and efficient than blood testing to identify infected herds. Results indicated that it could be advisable that legislation, rather than defining very detailed procedures both for laboratory tests and testing programmes, should establish minimal requirements in terms of efficacy of testing procedures (i.e. the probability of detecting an infected herd).

Kinetics of the antibody response in ewes experimentally infected with Brucella melitensis biovar 3.

The authors evaluated the kinetics of antibody response in 46 ewes coming from officially brucellosis free flocks that were experimentally infected with Brucella melitensis biovar 3, and monitored through three subsequent reproductive cycles. In this study, results of Rose Bengal test (RBT) and complement fixation test (CFT) were considered. Test results of 2nd and 3rd reproductive cycle show a peak in the antibody production at parturition, followed by a drop in the following months. The peak at parturition is significantly lower in the 3rd reproductive cycle compared to the 2nd. The drop in antibody production observed after parturition of the 3rd reproductive cycle is significantly higher than that observed after parturition of the 2nd reproductive cycle. Nevertheless, the infection can still be revealed at flock level after three years post infection.

Quality assurance of veterinary services at the international level: a proposed approach.

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.

Quality assurance in veterinary diagnostic laboratories.

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.

Assessment of an indirect ELISA in milk for the diagnosis of ovine Brucellosis.

The possibility of using an ELISA for the diagnosis of ovine brucellosis in milk (M-ELISA) was investigated. The aim of the study was to establish whether the specificity and sensitivity of the M-ELISA would be high enough to detect low levels of Brucella antibodies in ewe milk. The diagnostic performances of the test under study were established by means of reference standards and compared with conventional screening and confirmatory tests under field conditions. The diagnostic specificity of the M-ELISA established on a number of samples from Brucella-free flocks was 100% while relative to RBT and CFT positive reactors the M-ELISA demonstrated sensitivity of 65 and 83% respectively. Its sensitivity relative to culture positive animals was of 92%. The course of Brucella antibodies in milk of positive sheep was evaluated in colostrum and in mature milk for a period of 30 days after delivery and it appeared that concentrations of immunoglobulins in milk tend to sharply decrease soon after parturition while in blood serum these remained constantly high. It was concluded that the M-ELISA for Brucella antibodies in ewe milk can be regarded as a complementary diagnostic tool for individual testing but it would be poorly viable if used as a screening test applied to pooled flock milks.

Sero-prevalences of selected cattle diseases in the Kafue flats of Zambia.

Sera from five traditionally managed herds grazing in the Kafue flats were tested for antibodies to bovine viral diarrhoea-mucosal disease (BVD-MD), parainfluenza 3 (PI3), infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (IBR-IPV), bovine adenovirus 3 (BAV3) and Bluetongue (BT). The sero-prevalences of the first four diseases were respectively 76.2, 94.4, 42.1 and 87.4%. Five samples (2.3%) gave doubtful reactions for BT. Prevalences of 28.5% for brucellosis, 14% for Rift Valley fever (RFV), 0.9% for Q fever and 11.2% for chlamydiosis were also recorded. Significantly higher values for BVD-MD (p less than 0.005), IBR-IPV (p less than 0.01) and brucellosis (p less than 0.05) were found in animals over 1 year of age. No differences were recorded between herds or between male and female animals. The high concentration of wild and domestic ruminants grazing together in the flood plains during the dry season may be a major determinant of the high values observed. Traditional farmers, slaughterhouse workers and other people involved in livestock production are particularly at risk of contracting brucellosis and RVF because of the high prevalences in cattle and local habits favourable to their transmission.

Noninvolvement of gastric mucus in the mucolytic effect of stepronine lysine salt.

The possible influence of stepronine lysine salt, a mucolytic agent, on human gastric mucus was investigated in ten healthy volunteers. Stepronine was administered by the oral route for ten days at a dose of 810 mg daily. Neither the total amount of mucoproteins in the gastric juice nor the quality of gastric mucus secretion (as assessed by a 'mucoprotective index') were altered after treatment with the bronchial secretolytic. The results indicate that the mucolytic activity of stepronine does not involve the gastric mucous coating and that the drug does not exert adverse effects on the gastric mucosa.