Molecular characterization of Italian bovine leukemia virus isolates reveals the presence of distinct phylogenetic clusters.

Disease caused by bovine leukemia virus (BLV) results in significant economic losses to the livestock industry. To date, there is only one report describing the strains found in Italy. BLV strains (n = 24), collected between 2012 and 2016 from four different Italian regions, were genetically analyzed by direct sequencing of a portion of the BLV env gene, and the sequences were compared with those in the GenBank database. The Italian BLVs clustered into genotypes G2, G4, G6, G7, and G8, revealing a high level of BLV genetic heterogeneity in Italy. This study provides a basis for further investigations into the evolutionary relationship between BLV strains.

Biological Containment for African Swine Fever (ASF) Laboratories and Animal Facilities: The Italian Challenge in Bridging the Present Regulatory Gap and Enhancing Biosafety and Biosecurity Measures.

The African Swine Fever Virus (ASFV) is a DNA virus of the Asfarviridae family, Asfivirus genus. It is responsible for massive losses in pig populations and drastic direct and indirect economic impacts. The ever-growing handling of ASFV pathological material in laboratories, necessary for either diagnostic or research activities, requires particular attention to avoid accidental virus release from laboratories and its detrimental economic and environmental effects. Recently, the Commission Delegated Regulation (EU) 2020/689 of 17 December 2019 repealed the Commission Decision of 26 May 2003 reporting an ASF diagnostic manual (2003/422/EC) with the minimum and supplementary requirements for ASF laboratories. This decision generated a regulatory gap that has not been addressed yet. This paper aims to describe the Italian National Reference Laboratory (NRL) efforts to develop an effective and reliable biological containment tool for ASF laboratories and animal facilities. The tool consists of comprehensive and harmonized structural and procedural requirements for ASF laboratories and animal facilities that have been developed based on both current and repealed legislation, further entailing a risk assessment and internal audit as indispensable tools to design, adjust, and improve biological containment measures.

Strategic Challenges to the Eradication of African Swine Fever Genotype II in Domestic Pigs in North Italy.

African swine fever (ASF) is a severe viral disease characterized by high lethality in suids and caused by the African Swine Fever Virus (ASFV). The ASF genotype I virus was introduced to Europe in 1957, marking the onset of the first European epidemic wave. In 2007, ASFV genotype II was detected in Georgia, affecting domestic pigs and wild boars before spreading to various European and extra-European countries, including Italy. The first case of ASFV in Italy was documented on 7 January 2022, in a wild boar in the Piedmont region. Since then, several ASFV-positive wild boar carcasses have been identified in the Piedmont and Liguria regions. By June 2023, ASFV had spread to Lombardy, one of the major pig-producing regions in northern Italy; the virus was first detected in early summer in wild boar carcasses. Two months later, it was diagnosed in a commercial pig farm as a consequence of the disease's spread amongst wild boars and an increase in the viral environmental load. This report aims to describe the features of ASFV domestic pig outbreaks that occurred in the Zinasco municipality (Lombardy) and the joint efforts to mitigate potential direct and indirect economic impacts on the Italian and global pig industry. The epidemiological investigation and the measures implemented, which were all performed according to national and European regulations, as well as exceptional ad hoc measures aimed at protecting the pig industry, are described in order to provide a practical and effective approach to combating ASF.

Using scenario tree modelling to evaluate the probability of freedom from Enzootic bovine leukosis (EBL) in Italy and Slovenia.

Documented freedom from disease is paramount for international free trade of animals and animal products. This study describes a scenario tree analysis to estimate the probability of freedom from Enzootic bovine leukosis (EBL) in Italy and Slovenia using information gathered via the data collection tool developed in the COST action project SOUND-control. Data on EBL control programmes (CPs) from 2018 to 2021 were used to build the models. Since animals are only sampled on the farm, one surveillance system component (SSC) was considered. The posterior probability of freedom (PostPfree) was estimated in time steps of one year, from 2018 to 2021.  After each year, the calculated from the previous year, combined with the probability of introduction, was used as a prior probability for the next year.  The herd level design prevalence was set to 0.2% in accordance with the Council Directive 64/432/EEC and the within herd design prevalence was set to 15%.  As Slovenia implemented a risk-based surveillance, targeting the herds importing cattle, in its model the design herd prevalence was combined with an average adjusted risk to calculate the effective probability of a herd importing cattle being infected.  The models were run for 10,000 iterations.  Over the study period the mean estimates were: i) for Italy both the surveillance system sensitivity ( SSe) and PostPFree 100%, with no differences between simulations and years, ii) for Slovenia the SSe was 50.5% while the PostPFree was 81.6%.

Genetic Characterization of African Swine Fever Italian Clusters in the 2022-2023 Epidemic Wave by a Multi-Gene Approach.

The first report of African swine fever virus (ASFV) genotype II in Italy in 2022 marked the beginning of a significant invasion in at least eight Italian regions with different infection clusters. In this study, we used the multi-gene approach to investigate the epidemiological associations between ASFV strains causing cases and outbreaks in wild boar and pigs in Italy from January 2022 to the end of 2023. Our results confirm that all the tested ASFV-positive Italian samples belonged to genotype II and show high homology with genotype II ASFV sequences previously collected in Eurasian countries. Molecular characterization revealed the presence of four genetic groups in Italy. The majority of African swine fever (ASF) samples analyzed in the current study (72%) belonged to genetic group 3, which was the most representative in Europe. The results also provide evidence of the prevalence of genetic group 19 (15.9%). In addition, we identified new putative genetic groups, genetic group 25 (9.1%) and genetic group 26 (3.0%), which have never been described before. This is the first detailed report on the molecular characterization of more than 130 ASFV strains circulating in Italy.

African Swine Fever: The State of the Art in Italy.

African swine fever (ASF) is a severe viral disease of domestic pigs and Eurasian wild boars (Sus scrofa) caused by the African swine fever virus (ASFV). ASF is endemic in sub-Saharan Africa, where 24 genotypes of the virus have been reported. Between the late 1950s and the early 1980s, genotype I ASFV emerged in Europe, including Italy. In June 2007, a second ASF epidemic wave caused by genotype II was registered, involving several European and extra-European countries, including Italy in 2022. The present paper aims to provide the state of the art of ASF in Italy, describing the course of ASF in wild boars and domestic pigs as an example of multiple concurring different scenarios. Sardinia is coping with the last phase of the eradication of the disease by applying the exit strategy. Conversely, four clusters of infection located in North, Central, and South Italy are still ongoing. The unique and complex Italian experience in ASF-controlling may be useful to increase know-how on the efficacy of strategies and measures, as well as issues that could be further improved.

Molecular Characterization of the First African Swine Fever Virus Genotype II Strains Identified from Mainland Italy, 2022.

African swine fever (ASF) is responsible for important socio-economic effects in the global pig industry, especially for countries with large-scale piggery sectors. In January 2022, the African swine fever virus (ASFV) genotype II was identified in a wild boar population in mainland Italy (Piedmont region). This study describes the molecular characterization, by Sanger and next-generation sequencing (NGS), of the first index case 632/AL/2022 and of another isolate (2802/AL/2022) reported in the same month, in close proximity to the first, following multiple ASF outbreaks. Phylogenetic analysis based on the B646L gene and NGS clustered the isolates 632/AL/2022 and 2802/AL/2022 within the wide and most homogeneous p72 genotype II that includes viruses from European and Asian countries. The consensus sequence obtained from the ASFV 2802/AL/2022 isolate was 190,598 nucleotides in length and had a mean GC content of 38.38%. At the whole-genome level, ASF isolate 2802/AL/2022 showed a close genetic correlation with the other representative ASFV genotype II strains isolated between April 2007 and January 2022 from wild and domestic pigs in Eastern/Central European (EU) and Asian countries. CVR subtyping clustered the two Italian ASFV strains within the major CVR variant circulating since the first virus introduction in Georgia in 2007. Intergenic region I73R-I329L subtyping placed the Italian ASFV isolates within the variant identical to the strains frequently identified among wild boars and domestic pigs. Presently, given the high sequence similarity, it is impossible to trace the precise geographic origin of the virus at a country level. Moreover, the full-length sequences available in the NCBI are not completely representative of all affected territories.

Complete Genome of African Swine Fever Virus Genotype II in Central Italy.

We report here the whole-genome sequence of the African swine fever virus (ASFV) genotype II, strain 20355/RM/2022_Italy, identified in a wild boar in the city of Rome (Lazio region, Italy) in April 2022.

Disinfectants against African Swine Fever: An Updated Review.

African Swine Fever (ASF), a hemorrhagic disease with a high mortality rate in suids, is transmitted via direct and indirect contact with infectious animals and contaminated fomites, respectively. ASF reached Europe in 2014, affecting 14 of the 27 EU countries including, recently, the Italian peninsula. The fast and unprecedented spread of ASF in the EU has highlighted gaps in knowledge regarding transmission mechanisms. Fomites, such as contaminated clothing and footwear, farming tools, equipment and vehicles have been widely reported in the spread of ASF. The absence of available vaccines renders biosecurity measures, cleaning and disinfection procedures an essential control tool, to a greater degree than the others, for the prevention of primary and secondary introductions of ASF in pig farms. In this review, available data on the virucidal activity of chemical compounds as disinfectants against the ASF virus (ASFV) are summarized together with laboratory methods adopted to assess the virucidal activity.

Analysis of surveillance and prevention plan for African Swine Fever in Italy in 2020.

BACKGROUND: African Swine Fever (ASF) is a challenge for pig health worldwide. The disease has spread to multiple countries on five continents. ASF-free countries need to apply effective strategies to prevent the introduction of infection. METHODS: Italy implemented a surveillance and prevention plan for ASF in 2020, supported by a dedicated information system. Several pillars for action have been identified: passive surveillance in both domestic pigs and wild boar populations, verification of the implementation of biosecurity measures on farms and an awareness campaign for all involved stakeholders. RESULTS: There were some regional differences in the management of passive surveillance. In order to identify all critical points and apply corrective measures, regional authorities were called to carry out a gap analysis exercise in July 2020. There were an adequate number of samples collected from wild boar but the number of samples collected from domestic pigs was below the target in most regions. Furthermore, sample distribution within the country was not homogeneous. CONCLUSIONS: During the forthcoming year, some issues must be addressed in order to establish an effective early detection system in Italian ASF-free regions.

Validation of a Commercial Indirect ELISA Kit for the Detection of Bovine alphaherpesvirus1 (BoHV-1)-Specific Glycoprotein E Antibodies in Bulk Milk Samples of Dairy Cows.

In this study, we validated a commercial indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies to glycoprotein E (gE) of Bovine alphaherpesvirus 1 (BoHV-1) in bulk milk (BM) samples using the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. The assay performance characteristics were evaluated using a panel of positive (n = 36) and negative (n = 80) samples with known infectious bovine rhinotracheitis (IBR) status. The assay showed adequate repeatability (within-run and between-run), with a coefficient of variability (CV%) of replicates below 30%; only two 1:40 diluted samples had a CV% above 20%. Additionally, an agreement analysis of the qualitative results of replicates led to a Gwet's agreement coefficient of 0.99 (95% confidence interval (CI): 0.96−1.00, p < 0.001). The estimated diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were 100% (95% CI: 90.3−100%) and 97.5% (95% CI: 91.3−99.7%), respectively. Overall, a good level of agreement was observed between the assay results and the true IBR status of samples (weighted Cohen's κ: 0.96, 95% CI: 0.78−1.00). The findings demonstrate that the indirect ELISA kit validated here is an easy-to-use and economical method to differentiate infected and gE-deleted marker vaccine-immunised animals using BM samples.

Evaluation of Serological Methods and a New Real-Time Nested PCR for Small Ruminant Lentiviruses.

Small ruminant lentiviruses (SRLVs), i.e., CAEV and MVV, cause insidious infections with life-long persistence and a slowly progressive disease, impairing both animal welfare and productivity in affected herds. The complex diagnosis of SRLVs currently combines serological methods including whole-virus and peptide-based ELISAs and Immunoblot. To improve the current diagnostic protocol, we analyzed 290 sera of animals originating from different European countries in parallel with three commercial screening ELISAs, Immunoblot as a confirmatory assay and five SU5 peptide ELISAs for genotype differentiation. A newly developed nested real-time PCR was carried out for the detection and genotype differentiation of the virus. Using a heat-map display of the combined results, the drawbacks of the current techniques were graphically visualized and quantified. The immunoblot and the SU5-ELISAs exhibited either unsatisfactory sensitivity or insufficient reliability in the differentiation of the causative viral genotype, respectively. The new truth standard was the concordance of the results of two out of three screening ELISAs and the PCR results for serologically false negative samples along with genotype differentiation. Whole-virus antigen-based ELISA showed the highest sensitivity (92.2%) and specificity (98.9%) among the screening tests, whereas PCR exhibited a sensitivity of 75%.

Genetic characterisation of African swine fever viruses from recent and historical outbreaks in Sardinia (1978-2009).

Three discrete regions of the African swine fever virus (ASFV) were analysed in the genomes of a wide range of isolates collected from wild and domestic pigs in Sardinia, over a 31-year period (1978-2009). The analysis was conducted by genotyping based on sequence data from three single copy ASF genes. The E183L gene encoding the structural protein p54 and part of the gene encoding the p72 protein were used to delineate genotypes, before intra-genotypic resolution of viral relationships by analysis of tetramer amino acid repeats within the hypervariable central variable region (CVR) of the B602L gene. The data revealed that these isolates did not show significant variation in their p72 and p54 sequence when compared between different isolates showing a remarkable genetic stability of these genome regions. In particular, the phylogeny revealed that all the Sardinian isolates belong to the same largest and most homogeneous p72 genotype I together with viruses from Europe, South America, the Caribbean and West Africa, and p54 genotype Ia which comprises viruses from Europe and America. The analysis of B602L gene revealed a minor difference in the number of tetramer repeats, placing the Sardinian isolates into two clusters, accordingly to their temporal distribution, namely sub-group III and sub-group X, this latter showing a deletion of 12 tetramer repeats located in the centre of the array. The genetic variation of this fragment suggests that one sub-group could be derived from the other supporting the hypothesis of a single introduction of ASFV in Sardinia.

Control programs for infectious bovine rhinotracheitis (IBR) in European countries: an overview.

Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus 1 (BoHV-1), is a disease of cattle responsible for significant economic losses worldwide. IBR is under certain communitarian regulations. Every member state can approve its own national IBR control program for the entire territory - or part of it - and can demand additional guarantees for bovids destined to its territory; therefore, every member state can be officially declared as entirely or partly IBR-free. The aim of this review is to provide an overview of IBR control and eradication programs in European countries. BoHV-1 control schemes were first introduced in the late 1970s, mainly in Northern and Central Europe. Depending on the seroprevalence rate, control strategies rely on identification and removal of seropositive animals or the use of glycoprotein E (gE)-deleted marker vaccines in infected herds. The implementation of a novel law for disease eradication at the EU level and of a European IBR data flow could make the goal of IBR eradication in all European countries easier to achieve.

Enzootic Bovine Leukosis in Italy: Epidemiological Issues after Free Status Recognition and Measures Applied to Tackle the Last Persistent Clusters.

Enzootic Bovine Leukosis (EBL), caused by the bovine leukemia virus (BLV), has been eradicated in over 20 countries, most of which are in Western Europe. The European Commission, in 2017, declared Italy to be an officially EBL-free country by means of Commission Implementing Decision (EU) 2017/1910, despite the presence of some infection clusters located in four regions of Central-Southern Italy. As a consequence of persisting infection, the Italian Ministry of Health established specific eradication measures in these areas. In collaboration with the National Reference Laboratory for the Study of Ruminant Retroviral Infectious Diseases, the Ministry of Health employed data from the veterinary information system digital platform, combined with a gap analysis exercise, to monitor and verify the progress of control activities within infection clusters during the period 2018-2021. Our aim was to identify any remaining gaps and, consequently, specific measures to eliminate the factors favouring EBL persistence, on the basis of a description and analysis of the current data regarding epidemiological trends in Italian clusters. The final goal is to achieve the implementation of a less expensive surveillance plan in these areas, as well. The results of comprehensive analysis showed that the eradication activities had been effectively implemented by official local veterinary services, resulting in a drastic reduction of EBL outbreaks in most territories during the period 2018-2021.

Genomic Epidemiology and Heterogeneity of SRLV in Italy from 1998 to 2019.

Small ruminant lentiviruses (SRLV) are viruses that retro-transcribe RNA to DNA and show high rates of genetic variability. SRLV affect animals with strains specific for each host species (sheep or goats), resulting in a series of clinical manifestations depending on the virulence of the strain, the host's genetic background and farm production system. The aim of this work was to present an up-to-date overview of the genomic epidemiology and genetic diversity of SRLV in Italy over time (1998-2019). In this study, we investigated 219 SRLV samples collected from 17 different Italian regions in 178 geographically distinct herds by CEREL. Our genetic study was based on partial sequencing of the gag-pol gene (800 bp) and phylogenetic analysis. We identified new subtypes with high heterogeneity, new clusters and recombinant forms. The genetic diversity of Italian SRLV strains may have diagnostic and immunological implications that affect the performance of diagnostic tools. Therefore, it is extremely important to increase the control of genomic variants to improve the control measures.

Overview of Control Programs for Twenty-Four Infectious Cattle Diseases in Italy

The cattle industry is a major driving force for the Italian agricultural sector totalling about 5. 6 million heads for dairy and meat production together. It is particularly developed in the northern part of the country, where 70% of the whole Italian cattle population is reared. The cattle industry development in the rest of the country is hampered by the hard orography of the territories and a variety of socioeconomic features leading to the persistence of the traditional rural farming systems. The differences in the farming systems (industrial vs. traditional) also affect the health status of the farms. Whereas, Enzootic Bovine Leukosis (EBL) is almost eradicated across the whole country, in Southern Italy where Bovine Tuberculosis and Brucellosis are still present and Bluetongue is endemic due to the presence of the competent vector (Culicoides imicola), less investments are aimed at controlling diseases with economic impact or at improving farm biosecurity. On the other hand, with the eradication of these diseases in most part of the country, the need has emerged for reducing the economic burden of non-regulated endemic disease and control programs (CPs) for specific diseases have been implemented at regional level, based on the needs of each territory (for instance common grazing or trading with neighboring countries). This explains the coexistence of different types of programs in force throughout the country. Nowadays in Italy, among cattle diseases with little or no EU regulations only three are regulated by a national CP: Enzootic Bovine Leukosis, Bluetongue and Paratuberculosis, while Bovine Genital Campylobacteriosis and Trichomonosis are nationwide controlled only in breeding bulls. For some of the remaining diseases (Infectious Bovine Rhinotracheitis, Bovine Viral Diarrhea, Streptococcus agalactiae) specific CPs have been implemented by the regional Authorities, but for most of them a CP does not exist at all. However, there is a growing awareness among farmers and public health authorities that animal diseases have a major impact not only on the farm profitability but also on animal welfare and on the use of antibiotics in livestock. It is probable that in the near future other CPs will be implemented.

Corrigendum: Overview of Control Programs for Twenty-Four Infectious Cattle Diseases in Italy.

[This corrects the article DOI: 10.3389/fvets.2021.665607.].

Evaluation of Passive Immunity Induced by Immunisation Using Two Inactivated gE-deleted Marker Vaccines against Infectious Bovine Rhinotracheitis (IBR) in Calves.

Different types of vaccines against Infectious Bovine Rhinotracheitis (IBR) are commercially available. Among these, inactivated glycoprotein E (gE)-deleted marker vaccines are commonly used, but their ability to induce passive immunity is poorly known. Here, we evaluated the passive immunity transferred from dams immunised with commercial inactivated gE-deleted marker vaccines to calves. We vaccinated 12 pregnant cattle devoid of neutralising antibodies against Bovine alphaherpesvirus 1 (BoHV-1) and divided them into two groups with 6 animals each. Both groups were injected with a different inactivated gE-deleted marker vaccine administrated via intranasal or intramuscular routes. An additional 6 pregnant cattle served as the unvaccinated control group. After calving, the number of animals in each group was increased by the newborn calves. In the dams, the humoral immune response was evaluated before calving and, subsequently, at different times until post-calving day 180 (PCD180). In addition, the antibodies in colostrum, milk, and in serum samples from newborn calves were evaluated at different times until PCD180. The results indicated that inactivated glycoprotein E (gE)-deleted marker vaccines are safe and produce a good humoral immune response in pregnant cattle until calving and PCD180. Moreover, results showed that, in calf serum, passive immunity persists until PCD180.

Serological Cross-Reactivity Between Bovine alphaherpesvirus 2 and Bovine alphaherpesvirus 1 in a gB-ELISA: A Case Report in Italy.

In this study, we demonstrated for the first time in Italy, the serological cross-reactivity between Bovine alphaherpesvirus 2 (BoHV-2) and Bovine alphaherpesvirus 1 (BoHV-1). Five months after arriving at a performance test station in Central Italy, a 6-month-old calf, which was part of a group of 57 animals, tested positive for BoHV-1 in a commercial gB-ELISA test. It was immediately transferred to the quarantine unit and subjected to clinical observation and serological and virological investigations. During this period, the calf showed no clinical signs. The results from laboratory investigations demonstrated the presence of antibodies via competitive glycoprotein B (gB) ELISAs, indirect BoHV-1 ELISAs, and indirect BoHV-2 ELISAs. Furthermore, the plaque reduction assay provided evidence for the presence of antibodies only for BoHV-2, whereas the virus neutralization test showed negative results for both BoHV-1 and BoHV-5. These findings strongly suggest the occurrence of a serological cross-reactivity between BoHV-2 and BoHV-1. Interference of BoHV-2 antibodies in serological BoHV-1 diagnostics should be considered during routine IBR tests, especially when animals are kept in a performance test station.