Contents lists available at ScienceDirect Preventive Veterinary Medicine journal homepage: www.elsevier.com/locate/prevetmed Alternative methods to reduce the animal use in quality controls of inactivated BTV8 Bluetongue vaccines Mirella Luciani, Tiziana Di Febo*, Gaetano Federico Ronchi, Flavio Sacchini, Emanuela Rossi, Simonetta Ulisse, Chiara Di Pancrazio, Daniela Antonucci, Romolo Salini, Liana Teodori, Michele Podaliri Vulpiani, Manuela Tittarelli, Mauro Di Ventura Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario, 64100, Teramo, Italy ARTICLE INFO Keywords: Bluetongue virus serotype 8 Cytokines quantification Inactivated vaccine ELISA VP2 quantification 3R Quality controls ABSTRACT The acceptance of serology data instead of challenge for market release of new batches of commercial vaccine is under evaluation by regulatory agencies in order to reduce the use of animals and costs for manufacturers. In this study two vaccines for Bluetongue virus serotype 8 were submitted to quality controls required by the European Pharmacopoeia and tested on sheep in comparison with a commercial inactivated vaccine. Body temperature, antibody titres and viraemia of vaccinated and controls sheep were recorded. In addition IL4 and IFNγ in sera and supernatant derived from in vitro stimulation of blood cells were also quantified using two commercial ELISA kit. The outer-capsid protein VP2 contained in vaccine formulations was quantified using a home-made capture- ELISA. Results obtained indicates that in-lab evaluation of cell-mediated and humoral immune response are useful parameters to predict the efficacy of BTV inactivated vaccines avoiding the challenge phase required to release new batches of vaccines with proven clinical efficacy and safety. The correlation observed between serology data and VP2 protein concentration of final product could be useful in-process control to predict if a new vaccine batch of BTV must be discarded or released to the market. 1. Introduction Bluetongue virus (BTV) is the causative agent of bluetongue, a vector-transmitted disease of domestic and wild ruminants. BTV be- longs to the genus Orbivirus and family Reoviridae and has a double stranded RNA genome (World Organisation for Animal Health (OIE), 2014; Maan et al., 2007). An animal infected with BTV may show leucopenia, fever, facial oedema, ulceration of oral mucosa, congestion of tongue and coronary band, debility, decreased production and variable mortality (MacLachlan et al., 2009). However, clinical signs were observed only in sheep; no apparent signs were reported in cattle, buffalo and goats in spite of the high seroprevalence registered in those species (Sreenivasulu et al., 2004). To date, 27 serotypes have been identified (Belbis et al., 2017). The BTV8 northern European strain was responsible for one of the largest outbreaks in the history of bluetongue. The virus emerged in 2006 in an area between the Netherlands, Bel- gium, and northern Germany and then spread throughout the con- tinent, causing significant economic losses due to animal movement restrictions, high mortality in naive sheep flocks and, occasionally, also clinical disease in cattle (Saegerman et al., 2010; Savini et al., 2008; Schwartz-Cornil et al., 2008; Zientara et al., 2010). In Italy, the first evidence of BTV infection was recorded in Sardinia in 2000; in 2008 BTV8 infection was detected in northern Italy (Verona province) and a vaccination campaign was carried out on livestock located in Verona and Mantova provinces (Calistri et al., 2010). Bluetongue vaccination is applied to prevent clinical disease, to limit regional extension of BTV outbreaks, to reduce virus circulation, to allow regional or country eradication of the disease and to authorize safe movement of suscep- tible animals between BTV infected and free zones (Savini et al., 2008), depending on the epidemiological situation of the affected area and objective of the strategy. Following high economic losses caused by several BTV8 outbreaks and consequent animal movement restrictions imposed thereafter, a large-scale vaccination scheme was introduced in European Union member countries and Switzerland Wäckerlin et al. (2010). Inactivated vaccines represents the preferred choice today, due to residual viru- lence observed in modified-live BT vaccines used in the past (Feenstra and van Rijn, 2017). Safety and efficacy of every vaccine batch produced have to be verified before release to the market. Currently animal testing, such as https://doi.org/10.1016/j.prevetmed.2020.104923 Received 14 October 2019; Received in revised form 17 January 2020; Accepted 5 February 2020 ⁎ Corresponding author. E-mail address: t.difebo@izs.it (T. Di Febo). Preventive Veterinary Medicine 176 (2020) 104923 0167-5877/ © 2020 Elsevier B.V. All rights reserved. T