Development of two competitive ELISAs based on monoclonal antibodies for the serological detection of Bovine ephemeral fever virus.

Bovine ephemeral fever virus (BEFV) is a member of the genus Ephemerovirus in the family Rhabdoviridae. It is an arthropod-borne virus transmitted by many species of midges and mosquitoes. It can cause severe economic consequences due to losses in milk production and the general condition of cattle and water buffalo. BEF occurs in some tropical, subtropical and warm temperate regions of Africa, Australia, the Middle East and Asia with seasonal outbreaks, but its possible spread to other areas (e.g. Europe) cannot be excluded. Therefore, using and developing rapid diagnostic methods with optimal performance is essential for identifying emerging pathogens and their control. In the present study, we developed two competitive serological ELISAs based on monoclonal antibodies (mAbs), designed by using BEFV inactivated antigen and the BEF recombinant nucleoprotein (N), respectively. A panel of 77 BEF-positive and 338 BEF-negative sera was used to evaluate the two tests. With a diagnostic sensitivity of 97.4 % using the inactivated virus and 98.7 % using the recombinant N, and a diagnostic specificity of 100 % using both antigens, our results suggest that these tests are suitable for the serological diagnosis of BEF.

Molecular and Serological Detection of Bovine Coronaviruses in Marmots (Marmota marmota) in the Alpine Region.

In this study, virological surveillance focused on coronaviruses in marmots in the Alpine region in 2022, captured as part of a population control reduction program in the Livigno area. Seventy-six faecal samples were randomly collected from marmots at the time of capture and release and tested for genome detection of pan-coronavirus, pan-pestivirus, canine distemper virus, and influenza A and D virus. Nine faecal samples were positive in the Pan-CoV RT-PCR, while all were negative for the other viruses. Pan-coronavirus positives were further identified using Illumina's complete genome sequencing, which showed the highest homology with Bovine Coronavirus previously detected in roe deer in the Alps. Blood samples (n.35) were collected randomly from animals at release and tested for bovine coronavirus (BCoV) antibodies using competitive ELISA and VNT. Serological analyses revealed that 8/35 sera were positive for BCoV antibodies in both serological tests. This study provides molecular and serological evidence of the presence of BCoV in an alpine marmot population due to a likely spillover event. Marmots share areas and pastures with roe deer and other wild ruminants, and environmental transmission is a concrete possibility.

Isolation of Batborne Neglected Zoonotic Agent Issyk-Kul Virus, Italy.

We isolated Issyk-Kul virus (ISKV) from a bat sampled from Italy in 2021 and conducted ISKV-specific surveillance in bats collected in Italy during 2017-2023. ISKV circulation among synanthropic and sedentary species of bat, such as Savi's pipistrelle bat (Hypsugo savii) in northern Italy, may have public health implications in this region.

Detection and Molecular Characterization of Canine Distemper Virus in Wildlife from Northern Italy.

Canine distemper virus (CDV) is a fatal, highly contagious disease found in wild and domestic carnivores. Several outbreaks have occurred in wildlife in Italy in recent years. This study aims to detect CDV in wildlife following the increasing mortality of foxes (Vulpes vulpes) in the Emilia-Romagna region (northern Italy) observed in 2021. Sixty-seven foxes and one badger (Meles meles) were subjected to necropsy followed by histological examination and were analyzed with molecular techniques to detect the presence of CDV. Of the tested animals, 16% (nine foxes and one badger) were positive for CDV. Phylogenetic analysis showed two different lineages based on complete H gene sequences. The Europe/South America-1 lineage was detected in one fox from Modena, which resembled the CDV variant associated with a previous outbreak in northern Italy in 2018, while the European Wildlife lineage was detected in animals from the Rimini province. Amino acid analysis highlighted a Y549H mutation in all sequences collected, which is commonly associated with increased virulence.

FTA Cards as a Rapid Tool for Collection and Transport of Infective Samples: Experience with Foot-and-Mouth Disease Virus in Libya.

Foot-and-mouth disease (FMD) is a viral disease, widespread and highly contagious, that mainly affects cloven-hoofed domestic and wild animals. FMD can lead to high economic losses due to the reduction in animal production such as a drop in milk production, loss of body weight, and a high mortality rate in young ruminants. Sixteen samples were collected from animals showing typical clinical signs of FMD during the last FMD outbreak in Libya in 2018-2019. Flinders Technology Associates (FTA) cards impressed with blood, swabs, or vesicular epithelium samples were shipped to the WOAH FMD reference laboratory in Brescia, Italy, and tested for the detection of FMD viruses. Nucleic acids were extracted from the FTA cards, and molecular testing based on real-time RT-PCR assays was carried out, of which one was specifically designed for the detection of the FMD virus of serotype O, topotype O/East Africa-3 (O/EA-3), that was further confirmed by a sequence analysis of the VP1 gene. The phylogenetic analysis of the VP1 gene showed a nucleotide identity of more than 99% between the virus circulating in Libya and the FMD virus strains isolated in Algeria in 2019.

Retrieving Historical Cases of Aujeszky's Disease in Sicily (Italy): Report of a Natural Outbreak Affecting Sheep, Goats, Dogs, Cats and Foxes and Considerations on Critical Issues and Perspectives in Light of the Recent EU Regulation 429/2016.

Aujeszky's disease is caused by Suid alphaherpesvirus 1, and its main reservoir host is the pig. However, other species are also susceptible. Infection with this virus causes a severe neurological clinical picture named Aujeszky's disease, usually accompanied by itching and death a few days after the onset of symptoms. This study reports a multi-species outbreak of Aujeszky's disease that occurred in Sicily, which led to the death of 2 goats, 15 sheep, 2 dogs, 2 cats and 2 foxes. The diagnosis was made by culture, indirect immunofluorescence on brain samples and confirmed by biological test on rabbits. This study reports the first cases of Aujeszky's disease in Italy in cats, goat and sheep. The finding of Aujeszky's disease in several species in Sicily suggests a potential epizootic risk. In such areas where a multi-host system is recognised, an analysis of the risk factors should be carried out in order to develop targeted strategies for the control and eradication of the disease. The critical issues that hinder the control of Aujeszky's disease in the studied territory and perspectives for eradication in the light of EU regulation 429/2016 are also discussed.