Characterization of new small ruminant lentivirus subtype B3 suggests animal trade within the Mediterranean Basin.

Small ruminant lentiviruses (SRLVs) represent a group of viruses infecting sheep and goats worldwide. Despite the high heterogeneity of genotype A strains, which cluster into as many as ten subtypes, genotype B was believed to be less complex and has, so far, been subdivided into only two subtypes. Here, we describe two novel full-length proviral sequences isolated from Sarda sheep in two Italian regions. Genome sequence as well as the main linear epitopes clearly placed this cluster into genotype B. However, owing to long-standing segregation of this sheep breed, the genetic distances that are clearly >15 % with respect to B1 and B2 subtypes suggest the designation of a novel subtype, B3. Moreover the close relationship with a gag sequence obtained from a Turkish sheep adds new evidence to historical data that suggest an anthropochorous dissemination of hosts (small ruminants) and their pathogens (SRLV) during the colonization of the Mediterranean from the Middle East.

Risk factors associated with the occurrence of undesired effects in sheep and goats after field vaccination with modified-live vaccine against bluetongue virus serotypes 2, 4 and 16.

In the 2004, the Sardinian bluetongue (BT) vaccination campaign used the combination of monovalent BTV-2, BTV-4 and BTV-16 modified-live vaccines manufactured by the Onderstepoort Biological Products in South Africa. Following vaccination, some herds showed temperature, oedema, lameness, hyperaemia and decrease in milk production, and some others remained perfectly healthy. This study aimed to evaluate whether important factors present in the herd at the time of vaccination could be associated to the occurrence of undesired effects observed after immunisation with BTV modified-live vaccines. A sample of 17 sheep and 4 goat flocks, for a total of 670 animals, were included in the study and risk factors such as presence of most important parasitic, bacterial and viral diseases as well as anomalies of biochemical and haematological parameters were associated to the presence or absence of side effects. For each factor the relative risk and 95% confidence interval were calculated. Following vaccination, bluetongue-like symptoms were observed in 13 flocks. In these flocks, a higher (P<0.05) proportion of animals had viraemia and showed higher titers to BTV-16 after immunisation. Positive association (RR=2.50, 1.17-5.04) was also found between flocks in which undesired effect were observed and positive serology against Maedi-Visna virus. On the contrary, presence of BTV genome fractions in the blood of animals at the time of vaccination was found to be protective (RR=0.7, 0.58-0.84) to the occurrence of undesired effect subsequent to BTV vaccination.

Epidemiological study of Toxoplasma gondii infection in ovine breeding.

An outbreak of toxoplasmosis occurring in a typical farm of 524 ovines was monitored for 1 year after the occurrence of 31 abortions. Abortion events involved 7.2% of 430 pregnant sheep. Presence of antibodies to Toxoplasma gondii in sheep sera was investigated by the indirect fluorescence antibody test (IFAT). A total of 422 ewes were bled four times during the year, and an epidemiological analysis was performed on all serology data collected in this subgroup. The prevalence of IgG positives ranged from 31.52% (133/422) at the first sampling to 62.56% (264/422) at the fourth sampling. Incidence of IgG antibodies was 38.75% at the second sampling, 14.92% at the third and 29.28% at the fourth sampling. At the beginning of the study, prevalence was 70.7% in primiparous sheep and 20.9% in sheep older than 5 years; at the third sampling, prevalence was stable at 70% in pluriparous sheep. The mean prevalence of IgM antibodies was 14.87%. A total of 147 out of all 524 ovines of the flock tested positive for IgM in more than one sampling. After an initial positivity, 60 sheep tested negative for IgG at the following serological controls (4 between the first and the second sampling, 30 between the second and the third and 28 between the third and the fourth sampling). One stray cat was positive for IgG, with a titre of 1 : 320. Moreover, one of the farmers was also positive, with a titre of 1 : 160 for IgG. A positive PCR result for T. gondii DNA was also observed in aliquots of grain and pellets taken from feed stocks amassed inside the sheds without protection, suggesting that an adequate management of the farm might be useful, if not essential, for controlling T. gondii outbreaks in ovine flocks.

p.Asn176Lys and p.Met137Thr dimorphisms of the PRNP gene significantly decrease the susceptibility to classical scrapie in ARQ/ARQ sheep.

In this study, we investigated the susceptibility to scrapie of Sarda breed sheep carrying the genotype ARQ/ARQ with additional polymorphisms at the PRNP gene. To do this, we examined 256 scrapie-affected sheep and 320 flock-mate negative controls from 24 flocks. Logistic regression analysis demonstrated that sheep carrying the ARQ/ARQ genotype with additional dimorphisms had lower risk of becoming scrapie affected when compared with those with ARQ/ARQ(wildtype) genotype. ARQ/ARQ genotypes that were detected with heterozygous or homozygous p.Asn176Lys and p.Met137Thr dimorphisms were associated with the lowest susceptibility to the disease. A significant lower risk was also associated with the p.Arg154His dimorphism, while p.Leu141Phe had a protective effect that was not statistically significant.

Two cases of human granulocytic ehrlichiosis in Sardinia, Italy confirmed by PCR.

In this work we report the first two cases of human granulocytic ehrlichiosis (HGE) in Sardinia. In early September 2004, a 69-year-old woman (patient 1) was admitted to the Infectious Diseases Institute of Sassari for rickettsiosis like-syndrome: high fever (39.5-40 degrees C), dyspnea, reduced consciousness, vomiting, and cutaneous rash. In late September 2004, a 30-year-old man (patient 2) with high fever was admitted for an evident palmar and oral erythema, edema of the labium, very intense arthralgia, myalgia, and dyspnea. In these two hospitalized patients, the diagnosis was made through indirect IgM and IgG immunofluorescent technique and confirmed by the presence of the specific DNA in the leukocytes. The two patients were A. phagocytophilum-PCR positive.

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.

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.

Efficacy and safety studies on an inactivated vaccine against bluetongue virus serotype 2.

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.

Temporal and spatial patterns of African swine fever in Sardinia.

Temporal patterns and spatial distribution of African swine fever (ASF) were studied through the analysis of routinely collected data in the ASF-endemic area of the Province of Nuoro, Sardinia. During 1993-1996, ASF outbreaks were reported from 45 out of the 82 municipalities of the study area. Overall farm-level incidence rate (IR) was 1.3 outbreaks per 100 farms-year. ASF peaked in 1995 (IR = 1.8) and declined in 1996 (IR = 0.82). Significant (P < 0.05) spring peaks of ASF outbreaks and affected municipalities were detected using statistical methods for circular distributions. Spatial clustering of ASF-affected municipalities, as evaluated by join-count statistics, was significant in 1993 (Zjc = -3.0, P < 0.01) and 1994 (Zjc = -3.2, P < 0.01) but not in 1995 (Zjc = -0.6, P = 0.55) and 1996 (Zjc = -1.2, P = 0.23). Extensive pig farming and ASF were spatially co-distributed (kappa = 0.51, 95% CI = 0.33-0.70).

Effect of husbandry methods on seropositivity to African swine fever virus in Sardinian swine herds.

Multiple logistic regression was used on serological data collected in the context of the Sardinian African swine fever (ASF) eradication program from pig farms in the province of Nuoro, Sardinia. The monthly percentage of ASFV-positive herds decreased significantly from October 1994 through March 1996 (P < 0.001). The farm-level risk of seropositivity to African swine fever virus (ASFV) was higher in free-range farms than in partial-confinement farms (odds ratios (OR) varied between 4.9 in October 1994, and 5.7 in March 1996, P < 0.001). The risk of infection for total-confinement farms was one-fifth of the risk for partial-confinement farms in October 1994 (OR = 0.2, P < 0.001), whereas in March 1996, the estimated OR was 0.57 and not significant (upper confidence limit = 1.1). The maintenance of ASFV in Sardinia was primarily associated with free-range pig farms. The natural logarithm of the number of pigs tested per visit in a farm was positively associated with the risk of herd seropositivity (OR = 2.6, P < 0.001).

Epidemiology of classical swine fever in Sardinia: a serological survey of wild boar and comparison with African swine fever.

A serological survey was carried out to establish the distribution of classical swine fever among wild boar in Sardinia, where that disease and African swine fever have been endemic in free-ranging domestic pigs and wild boar living in the mountainous areas of the province of Nuoro for several years. Blood samples were collected from 4752 wild boar shot during the period December 1988 to January 1992. An overall prevalence of 11 per cent was observed and the almost constant rate of about 9.8 per cent detected in the past three years indicates that the infection is well established. Wild boar seropositive to classical swine fever were found not only in the areas of the province of Nuoro where they share their habitat with free-ranging domestic pigs but also in other areas of the island where contacts between wild and domestic pigs are unlikely to occur. Therefore, transmission from wild boar to wild boar seems to play an important role in the spread and persistence of classical swine fever virus. In contrast, African swine fever virus is probably unable to persist in the wild boar population in the absence of the risk factor represented by their cohabitation with domestic free-ranging pigs infected with African swine fever.