Veterinary Microbiology 235 (2019) 180–187 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Genotyping of Pestivirus A (Bovine Viral Diarrhea Virus 1) detected in faeces T and in other specimens of domestic and wild ruminants at the wildlife- livestock interface ⁎ Sara Riccia,1,2, Sofia Bartolinia,1,2, Federico Morandib, Vincenzo Cuteria, Silvia Preziusoa, a School of Biosciences and Veterinary Medicine, University of Camerino, Italy b National Park of Monti Sibillini, Visso, MC, Italy ARTICLE INFO ABSTRACT Keywords: Pestiviruses are widespread in the world among ungulates and infect both domestic and wild animals causing Pestivirus A severe economic losses in livestock. Bovine Viral Diarrhea Virus type 1 (BVDV-1), now re-designated as Pestivirus BVDV-1 A, causes diseases mainly in cattle, while few data are available about infection in wild ruminants and about the Genetic typing role of these animals in viral maintenance and spread. In order to investigate BVDV-1 infection in domestic and Domestic and wild ruminants wild ruminants, especially at the wildlife/livestock interface, bulk tank milk from dairy cattle and sheep and Faeces spleen from red deer, roe deer and fallow deer were analysed. Furthermore, faecal samples from Apennine chamois and from wild deer were evaluated as a suitable sample for detecting and genotyping pestiviruses. BVDV-1 RNA was found in all animal species tested but not sheep. Genotyping based on partial 5′UTR and Npro sequences detected BVDV-1a in samples from Apennine chamois, red deer, roe deer and pasture-raised cattle, while BVDV-1c was found in a faecal sample from Apennine chamois and in a spleen sample from roe deer. For the first time BVDV-1 RNA was found and genotyped from faecal samples of wild ruminants and of cattle.BVDV- 1a detection in Apennine chamois, red deer, roe deer and pasture-raised cattle suggests that the eventuality of viral transmission at the wildlife/livestock interface should be carefully evaluated. BVDV subgenotype 1c was found for the first time in roe deer and Apennine chamois in Central Italy, therefore the epidemiological roleof these animals and the viral ecology should be further investigated. 1. Introduction used for phylogenetic analyses and genotyping of BVDV isolates, al- lowing detection of at least 21 different BVDV-1 subtypes named from Pestiviruses are globally distributed members of the family BVDV-1a to BVDV-1 u (Yeşilbağ et al., 2017). Flaviviridae that cause severe economic losses in livestock (Smith et al., Pestiviruses have a very broad host range, are widespread among 2017; Yeşilbağ et al., 2017). The most common species of the genus ungulates and infect both domestic and wild swine and ruminants, Pestivirus are Bovine Viral Diarrhea Virus type 1 (BVDV-1) and type 2 mostly in the families Bovidae and Cervidae (Ridpath and Neill, 2016). (BVDV-2), Classical Swine Fever Virus (CSFV) and Border Disease Virus Epidemiological data of Pestivirus infections in European wild rumi- (BDV). On the basis of the most recent release of the International nants are limited. Severe outbreaks in the Southern chamois (Rupicapra Committee on Taxonomy of Viruses (ICTV) in 2018, these pestiviruses pyrenaica) caused by BDV have been reported in Spain starting from have been re-designated as Pestivirus A (original designation BVDV-1), 2001 (Marco et al., 2007, 2015). Serological investigations in Europe Pestivirus B (BVDV-2), Pestivirus C (CSFV) and Pestivirus D (BDV) (talk. showed that Pestivirus infections in wild ruminants are sporadic (Olde ictvonline.org/taxonomy/, last access 3rd February 2019). Genetic Riekerink et al., 2005; Fernández-Sirera et al., 2011; Casaubon et al., changes in pestivirus genomes are very frequent and different genomic 2012; Fernández-Aguilar et al., 2016). Only a few cases of BVDV-1 RNA regions have been used for classification and typing purposes. For ex- detection in European wild ruminants are described (Vilček and ample, partial 5′UTR and Npro sequences have been most frequently Nettleton, 2006; Casaubon et al., 2012; Fernández-Aguilar et al., 2016). ⁎ Corresponding author at: Via Circonvallazione 93/95, 62024, Matelica, MC, Italy. E-mail address: [email protected] (S. Preziuso). 1 Ricci Sara and Bartolini Sofia contributed equally to this work. 2 Present affiliation of S. Ricci is Institute of Animal Nutrition and Functional Plant Compounds, University of Veterinary Medicine, Vienna, Austria, andS.Bartolini is a Vet Practitioner, Macerata, Italy. https://doi.org/10.1016/j.vetmic.2019.07.002 Received 15 April 2019; Received in revised form 4 July 2019; Accepted 6 July 2019 0378-1135/ © 2019 Elsevier B.V. All rights reserved. S. Ricci, et al. Veterinary Microbiology 235 (2019) 180–187 In order to detect and genotype BVDV, blood samples are usually (RecoverAll Total Nucleic Acid Isolation Kit, Ambion, Life analysed, but viral sequences have been obtained also from bulk milk Technologies). All procedures were carried out in a biosafety cabinet specimens (Luzzago et al., 2014; Vilček et al., 2001) and from spleen of with an ultraviolet lamp; a specific laboratory cleaner (DNA cleaner, dead animals (Nelson et al., 2016; Passler et al., 2009). Diatech pharmacogenetics) was used to provide effective cleaning from In Central Italy, the wild ruminants’ population is represented DNA and RNA. To avoid any contamination, no more than five samples mainly by roe deer (Capreolus capreolus), fallow deer (Dama dama), and were processed at the same time, and mono use plastic tools were used. sporadically by red deer (Cervus elaphus) and Apennine chamois A Pestivirus-negative bovine faecal sample was used as negative control (Rupicapra pyrenaica ornata)(Carnevali et al., 2009). The Apennine during each RNA extraction and RNA retrotranscription run. chamois is a subspecies of chamois currently living only in limited areas of Central Italy and it is classified as “vulnerable” in the red listof 2.2.1. Faecal samples preparation threatened species drawn up by the International Union for Conserva- Six millilitres of RNAse-free PBS were added to 1.5 g of each faecal tion of Nature (IUCN, 2018). The Apennine chamois is a protected sample. After mechanical disruption and mixing, samples were cen- species under Italian and international laws (L.157/92, CITES Appendix trifuged at 500 x g for 10 min at 4 °C; 1 ml of the supernatant was II) and reintroduction projects have been carried out in several pro- transferred to a new tube and was centrifuged at 13,000 x g for 15 min tected areas in Central Italy, according to the Italian Action Plan for the at 4 °C. The final supernatant (100 μl) was used for RNA extraction. In Apennine chamois. Starting from 2008, Apennine chamois has been case of pool samples (groups A and C), 200 μl of the final supernatant of reintroduced in the Monti Sibillini National Park, with further support each sample were mixed with the other samples included in the pool from the European project LIFE Coornata - development of coordinated and 100 μl of this mixture were used for RNA extraction. protection measures for Apennine chamois LIFE09 NAT/IT/000183 (years 2010–2014) (Rossetti et al., 2015). In the Monti Sibillini Na- 2.2.2. Bulk tank milk samples tional Park breeding of cattle, sheep and goats often relies on traditional Aliquots of 45 ml of each bovine or ovine bulk tank milk sample practice based on small farms and extensive farming systems, where were centrifuged at 500 x g for 20 min at 10 °C. All cream and the upper animals graze freely on mountain pastures. Vaccination rate for pesti- part of the liquid were discarded. The remaining whey was transferred viruses in livestock in this context is usually low and the risk of viral to a new tube and 2 ml were put back in the original tube together with transmission at the wildlife/livestock interface should be contemplated. the pellet. After mixing, each sample was transferred in a new tube and Considering that the Monti Sibillini National Park is a protected area centrifuged at 13,000 x g for 10 min. Once all the supernatant was where hunting is prohibited and that capture for collecting blood is discarded, the pellet was washed with RNAse-free PBS three times and rare, expensive and stressful for wild ruminants, alternative sampling used for RNA extraction (Supplementary file S1). methods should be evaluated. The aims of this work were i) to evaluate whether faeces can be used 2.2.3. Spleen samples to detect Pestivirus RNA in wild ruminants and ii) to investigate and Spleen samples were collected from hunters and were stored at characterise genetically pestiviruses infecting domestic and wild ru- −20 °C up to 1 month or at 4 °C up to 1 week before being brought to minants, with special attention to the wildlife/livestock interface. the laboratory. Aliquots of 50 mg of spleen were used for RNA extrac- tion by using 1 ml Trizol RNA Reagent (Thermo Fisher Scientific, 2. Materials and methods Massachussets, USA) following the manufacturer's instructions. After drying, the RNA pellet was resuspended in 20 μl RNAse-free water. 2.1. Study design and sampling 2.3. RNA retrotranscription Samples were obtained from different domestic and wild ruminants living in different areas of Marche Region (Central Italy), between 2014 Two microlitres of each RNA sample were retrotranscripted and 2016 (Tables 1 and 2, Fig. 1). Different specimens were taken from (PrimeScript RT reagent Kit, Takara Bio Inc.) in 20 μl final volume. The different groups of animals, preferring specimens that can be easily cDNA obtained was immediately used for PCR or stored at −20 °C. sampled. Samples were collected and grouped as follows (Table 3): faeces from wild ruminants (red deer and Apennine chamois) living in 2.4.