Since February 2020, Italy has been seriously affected by the SARS-CoV-2 pandemic. To support the National Health Care system, naso-pharyngeal/oropharyngeal swabs collected from suspected cases of Teramo province, Abruzzo region, are tested at Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise G. Caporale, for the presence of SARS-CoV-2 RNA. Out of 12,446 tested individuals, 605 returned positive results at least once, with prevalence significantly higher in men. A reduction of the level of viral RNA in the first swab per each positive patient collected over time was also observed. Moreover, 81 patients had at least one positive sample and two final negative tests: positivity in swabs lasted from 14 to 63 days, with a median value of 30 days. This shows the potential for the virus to coexist with patients for a long time, although we highlighted intermittent positivity in several cases. The evolution of the SARS-CoV-2 epidemiological situation and knowledge on viral shedding should be closely monitored, to interpret the findings correctly and adjust accordingly the surveillance activities.

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7 of sensitivity, 98.1 of specificity, 93 of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohens kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease.

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

Background: Lumpy skin disease virus (Poxviridae family—Capripoxvirus genus) is the aetiological agent of LSD, a disease primarily transmitted by hematophagous biting, affecting principally cattle. Currently, only live attenuated vaccines are commercially available, but their use is limited to endemic areas. There is a need for safer vaccines, especially in LSD-free countries. This research aims to develop and test a safe and efficacious inactivated vaccine. Moreover, in this study, we used keyhole limpet hemocyanin (KLH) as a positive marker to distinguish infected from vaccinated animals (DIVA). Methods: Lumpy skin disease virus was propagated on primary lamb testis cells and Madin–Darby bovine kidney cells (PLT and MDBK, respectively), and four inactivated vaccines were produced. The vaccines differed from each other with the addition or not of KLH and in cells used for virus propagation. To evaluate the safety and immunogenicity, the vaccines and two placebos were administered to six groups comprising six male calves each, and antibody response was investigated using both an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization (SN) test. In addition, the LSD/γ-interferon test and KLH (IgM-IgG) ELISA were performed on the collected samples. Furthermore, the use of KLH allowed us to distinguish vaccinated animals in the ELISA results, without any interference on the strength of the immune response against the LSDV. Finally, the efficacy of one of four vaccines was investigated through a challenge, in which one group of vaccinated animals and one animal control group were infected with a live field strain of LSDV. Results: Four out of the six control animals showed severe clinical signs suggestive of LSD, and, therefore, were euthanized for overcoming the predetermined limit of clinical score. By contrast, the vaccinated animals showed only mild symptoms, suggesting a reduction in severe disease notwithstanding the incapability of the vaccine in reducing the virus shedding. Conclusion: The vaccines produced were safe and able to elicit both a humoral and a cellular immune response, characteristics that, together with the demonstrated efficacy, make our vaccine a good candidate for countering the LSD spread in disease-free countries, thus also facilitating disease containment throughout the application of a DIVA strategy.

The significance of Trypanosoma equiperdum as the causative agent of dourine cannot be understated, especially given its high mortality rate among equids. International movement of equids should be subject to thorough health checks and screenings to ensure that animals are not infected with Trypanosoma equiperdum. This involves the implementation of quarantine protocols, testing procedures, and the issuance of health certificates to certify the health status of the animals. Three proteins, the peptidyl-prolyl cis-trans isomerase (A0A1G4I8N3), the GrpE protein homolog (A0A1G4I464) and the transport protein particle (TRAPP) component, putative (A0A1G4I740) (UniProt accession numbers SCU68469.1, SCU66661.1 and SCU67727.1), were identified as unique to T. equiperdum by bioinformatics analysis. The proteins were expressed as recombinant proteins and tested using an indirect ELISA and immunoblotting test with a panel of horse positive and negative sera for dourine. The diagnostic sensitivity, specificity and accuracy of the i-ELISAs were 86.7%, 53.8% and 59.0% for A0A1G4I8N3; 53.3%, 58.7% and 57.9% for A0A1G4I464; and 73.3%, 65.0% and 66.3% for A0A1G4I740, respectively, while the diagnostic sensitivity, specificity and accuracy of immunoblotting were 86.7%, 92.5% and 91.6% for A0A1G4I8N3; 46.7%, 81.3% and 75.8% for A0A1G4I464; and 80.0%, 63.8% and 66.3% for A0A1G4I740. Among the three proteins evaluated in the present work, A0A1G4I8N3 provided the best results when tested by immunoblotting; diagnostic application of this protein should be further investigated using a greater number of positive and negative sera.

Bluetongue virus (BTV) is the etiologic agent of bluetongue (BT), a viral WOAH-listed disease affecting wild and domestic ruminants, primarily sheep. The outermost capsid protein VP2, encoded by S2, is the virion’s most variable protein, and the ability of reference sera to neutralize an isolate has so far dictated the differentiation of 24 classical BTV serotypes. Since 2008, additional novel BTV serotypes, often referred to as “atypical” BTVs, have been documented and, currently, the full list includes 36 putative serotypes. In March 2015, a novel atypical BTV strain was detected in the blood of asymptomatic goats in Sardinia (Italy) and named BTV-X ITL2015. The strain re-emerged in the same region in 2021 (BTV-X ITL2021). In this study, we investigated the pathogenicity and kinetics of infection of BTV-X ITL2021 following subcutaneous and intravenous infection of small ruminants. We demonstrated that, in our experimental settings, BTV-X ITL2021 induced a long-lasting viraemia only when administered by the intravenous route in goats, though the animals remained healthy and, apparently, did not develop a neutralizing immune response. Sheep were shown to be refractory to the infection by either route. Our findings suggest a restricted host tropism of BTV-X and point out goats as reservoirs for this virus in the field.