Western Bluetongue virus serotype 3 in Sardinia, diagnosis and characterization.

Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides-borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind-blown dissemination of infected midges. Here, we report the first identification of BTV-3 in Sardinia, Italy. BTV-3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV-3 SAR2018 was isolated on cell culture. BTV-3 SAR2018 sequence and partial sequences obtained by next-generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99-100% of nucleotide identity) to BTV-3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa.

Why is African swine fever still present in Sardinia?

African swine fever (ASF) is an infectious disease of swine that has been present in Sardinia since 1978. Soon after introduction of the disease, several control and eradication programmes were established with limited success. Some researchers attributed the persistence of the disease in central and eastern areas to certain socio-economic factors, the existence of some local and traditional farming practices (i.e., unregistered free-ranging pigs known as brado animals) and the high density of wild boar in the region. In the past, scarcity of swine data in Sardinia complicated the evaluation and study of ASF on the island. More complete, accurate and reliable information on pig farms has become available as a result of the most recent eradication programmes. Here, we perform statistical modelling based on these data and the known distribution of domestic pig and wild boar to identify the main risk factors that have caused ASF persistence in Sardinia. Our results categorized, identified and quantified nine significant risk factors, six of which have not been previously described. The most significant factors were the number of medium-sized farms, the presence of brado animals and the combination of estimated wild boar density and mean altitude above sea level. Based on these factors, we identified regions in eastern and central Sardinia to be at greatest risk of ASF persistence; these regions are also where the disease has traditionally been endemic. Based on these risk factors, we propose specific control measures aimed at mitigating such risks and eradicating ASF from the island.

Understanding African Swine Fever infection dynamics in Sardinia using a spatially explicit transmission model in domestic pig farms.

African swine fever virus (ASFV) has been endemic in Sardinia since 1978, resulting in severe losses for local pig producers and creating important problems for the island's veterinary authorities. This study used a spatially explicit stochastic transmission model followed by two regression models to investigate the dynamics of ASFV spread amongst domestic pig farms, to identify geographic areas at highest risk and determine the role of different susceptible pig populations (registered domestic pigs, non-registered domestic pigs [brado] and wild boar) in ASF occurrence. We simulated transmission within and between farms using an adapted version of the previously described model known as Be-FAST. Results from the model revealed a generally low diffusion of ASF in Sardinia, with only 24% of the simulations resulting in disease spread, and for each simulated outbreak on average only four farms and 66 pigs were affected. Overall, local spread (indirect transmission between farms within a 2 km radius through fomites) was the most common route of transmission, being responsible for 98.6% of secondary cases. The risk of ASF occurrence for each domestic pig farm was estimated from the spread model results and integrated in two regression models together with available data for brado and wild boar populations. There was a significant association between the density of all three populations (domestic pigs, brado, and wild boar) and ASF occurrence in Sardinia. The most significant risk factors were the high densities of brado (OR = 2.2) and wild boar (OR = 2.1). The results of both analyses demonstrated that ASF epidemiology and infection dynamics in Sardinia create a complex and multifactorial disease situation, where all susceptible populations play an important role. To stop ASF transmission in Sardinia, three main factors (improving biosecurity on domestic pig farms, eliminating brado practices and better management of wild boars) need to be addressed.

Spatio-temporal Analysis of African Swine Fever in Sardinia (2012-2014): Trends in Domestic Pigs and Wild Boar.

African swine fever (ASF) is a notifiable viral disease affecting domestic pigs and wild boars that has been endemic in Sardinia since 1978. Several risk factors complicate the control of ASF in Sardinia: generally poor level of biosecurity, traditional breeding practices, illegal behaviour in movements and feeding of pigs, and sporadic occurrence of long-term carriers. A previous study describes the disease in Sardinia during 1978-2013. The aim of this study was to gain more in-depth knowledge of the spatio-temporal pattern of ASF in Sardinia during 2012 to May 2014, comparing patterns of occurrence in domestic pigs and wild boar and identifying areas of local transmission. African swine fever notifications were studied considering seasonality, spatial autocorrelation, spatial point pattern and spatio-temporal clusters. Results showed differences in temporal and spatial pattern of wild boar and domestic pig notifications. The peak in wild boar notifications (October 2013 to February 2014) occurred six months after than in domestic pig (May to early summer 2013). Notifications of cases in both host species tended to be clustered, with a maximum significant distance of spatial association of 15 and 25 km in domestic pigs and wild boars, respectively. Five clusters for local ASF transmission were identified for domestic pigs, with a mean radius and duration of 4 km (3-9 km) and 38 days (6-55 days), respectively. Any wild boar clusters were found. The apparently secondary role of wild boar in ASF spread in Sardinia could be explained by certain socio-economic factors (illegal free-range pig breeding or the mingling of herds. The lack of effectiveness of previous surveillance and control programmes reveals the necessity of employing a new approach). Results present here provide better knowledge of the dynamics of ASF in Sardinia, which could be used in a more comprehensive risk analysis necessary to introduce a new approach in the eradication strategy.

Serological Surveillance and Direct Field Searching Reaffirm the Absence of Ornithodoros Erraticus Ticks Role in African Swine Fever Cycle in Sardinia.

African swine fever (ASF), one of the most important diseases of swine, has been endemic in the Italian island of Sardinia for more than 35 years. During these decades, several strategies and eradication efforts have been implemented in the island with limited success. Strong climatic and ecological similarities exist between Sardinia and one area of the Iberian Peninsula where Ornithodoros erraticus ticks were involved in the persistence of ASF from 1960 to 1995. This fact leads to the hypothesis that, potentially, Ornithodoros ticks could be also involved in the ASF cycle in Sardinia, thus accounting for some of the reoccurring ASF outbreaks in this island. Initial efforts aimed at detection of Ornithodoros ticks in Sardinia were performed during the 1970s/1980s with no positive results. Accordingly, the absence of Ornithodoros ticks in Sardinia has been generally accepted. However, since a new and reinforced ASF eradication programme has been recently launched in Sardinia, it is essential to clarify the presence and role of these soft ticks in the epizootiology of ASF in this island. For that purpose, 1767 porcine serum samples collected from all around the island (1261 from domestic and 506 from wild boar) were analysed by ELISA for antibodies to salivary antigens of Ornithodoros erraticus. In addition, Ornithodoros ticks were directly searched in a number of pig premises that have suitable habitats for these ticks and were located in areas repeatedly affected by ASF. Only one serum sample resulted positive in the serological assay, and no Ornithodoros ticks were collected in none of the premises. These results indicate that these soft tick species are not involved in the epizootic cycle of ASF in Sardinia and highlight the importance of controlling other risk factors still present in the island for effectively eradicate the disease.

Thirty-Five-Year Presence of African Swine Fever in Sardinia: History, Evolution and Risk Factors for Disease Maintenance.

Despite the implementation of control efforts and funds to fight against the disease, African swine fever (ASF) has been present in Sardinia since 1978. It has caused serious problems for both the industrial pig sector and the regional authorities in Sardinia, as well as the economy of Italy and the European Union, which annually supports the costly eradication programme. During this time, ASF has persisted, especially in the central-east part of Sardinia where almost 75% of the total outbreaks are concentrated. The Sardinian pig sector is clearly divided into two categories based on the specialization and industrialization of production: industrial farms, which represents only 1.8% of the farms in the island and non-professional holdings, which are comprised of small producers (90% of pig holdings have <15 pigs) and apply little to no biosecurity measures. Additionally, illegally raised pigs are still bred in free-ranging systems in certain isolated parts of the island, despite strict regulations. The illegal raising of pigs, along with other high-risk management practices (e.g., use of communal areas) are likely the primary reasons for endemic persistence of the virus in this area. The compensation provided to the farmers, and other aspects of the eradication programme have also negatively influenced eradication efforts, indicating that socio-cultural and economic factors play an important role in the epidemiology of ASF on the island. The aim of this study was to comprehensively review the evolution of the 35-year presence of ASF in Sardinia, including control measures, and the environmental and socio-economic factors that may have contributed to disease endemicity on the island. The present review highlights the need for a coordinated programme that considers these socio-economic and environmental factors and includes an assessment of new cost-effective control strategies and diagnostic tools for effectively controlling ASF in Sardinia.

MLVA as an Epidemiological Tool To Trace Back Brucella melitensis Biovar 1 Re-Emergence in Italy.

Brucellosis is an important zoonosis caused by Brucella spp., still prevalent in most areas of the world. Brucellosis control in animals is the key to protect humans. The knowledge of Brucella spp. prevailing genotypes in a territory represents an important epidemiological tool to formulate policies and strategies for disease control and to trace back the introduction of new strains previously considered as exotic. In the last years, multiple-locus variable number tandem repeat analysis (MLVA) has been proposed as complementary to classical biotyping methods. MLVA may add important information to the classical epidemiological investigation techniques, to help in tracing back sources of infection in brucellosis outbreaks. Sardinia is an Italian region officially free from sheep and goats brucellosis since 1998. In 2011, Brucella melitensis biovar 1, a biotype not reported in Italy since 1995, was isolated in one flock in the region. The genotyping MLVA-16 showed that isolates belonged to a rare American lineage, confirming it was introduced from other countries. The strain was considered as probably originating from Spain, where this lineage is endemic. BrucellaMLVA-16 has been proved to be useful to analyse the epidemiological correlation of strains enabling to trace its geographic origin by comparing their previously reported genetic patterns.

West Nile virus lineage 2 in Sardinian wild birds in 2012: a further threat to public health.

West Nile virus (WNV) strains belonging to lineage 2 were detected and isolated from the tissues of a goshawk and two carrion crows in Sardinia in August 2012. According to NS3 sequence analysis, the Sardinian isolates shared a high level of similarity with those of Italian lineage 2 strains which circulated in 2011 and with the homologous sequence of the 2004 Hungarian isolate. Following the human fatality reported in 2011 in Olbia, this study is the first to report the spread and enzootic circulation of WNV lineage 2 in Sardinia.

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.

Prevalence of Salmonella enterica and Listeria monocytogenes contamination in foods of animal origin in Italy.

The present survey collected and analyzed the results of routine testing for Salmonella enterica and Listeria monocytogenes on foods of animal origin submitted for official controls in Italy during 2001 to 2002. Salmonella was detected in 2.2% of 71,643 food samples examined, and the isolation rates ranged from 9.9% for raw poultry meat to less than 0.1% for dairy products. Isolation rates were also high in raw pork (4.9%) and processed meats (5.3%), which often involved pork. Low rates were observed in seafood (0.5%) and in ready-to-eat foods, such as grocery products (0.7%) and ice creams (0.1%). Serotyping showed that approximately 50% of the isolates belonged to the serotypes most commonly isolated from humans in Italy, thus confirming that most cases of human salmonellosis have a foodborne origin. Levels of L. monocytogenes were higher than what is accepted by the current regulation in 2.4% of 42,300 food samples. The positivity rates ranged from 10.3% in raw pork to none in eggs and egg products. Contamination rates were higher in other meat products (between 2 and 5%) and fish (6.5%) than in cheeses (1.1%) and other dairy products (0.6%). Routine control activities on the microbial contamination of foods can generate data with statistical and epidemiological value. Such data can be used as a basis for estimating the exposure of consumers to foodborne pathogens, following the trends of contamination over time, and evaluating the effects of control measures on the contamination of food.

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 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.