Long-term immunogenicity and protection against Mycoplasma agalactiae induced by an oil adjuvant vaccine in sheep Domenico Buonavoglia a, * , Grazia Greco a , Marialaura Corrente a , Maria Fiorella Greco a , Maria D’Abramo a , Francesca Latronico a , Antonio Fasanella b , Nicola Decaro a a Department of Veterinary Public Health, Faculty of Veterinary Medicine, University of Bari, Valenzano, Bari, Italy b Istituto Zooprofilattico Sperimentale della Puglia e Basilicata, Vaccines Production Center, Foggia, Italy article info Article history: Accepted 15 July 2009 Keywords: Mycoplasma agalactiae Sheep Oil-adjuvant vaccine Long-term protection abstract The long-term protective immunity of an inactivated mineral-oil adjuvanted Mycoplasma agalactiae vac- cine was evaluated in sheep. The antigen suspension was emulsified with a mixture of three mineral oils (Montanide ISA-563, Marcol-52, Montane-80 at the ratio of 30%, 63%, and 7%, respectively). Twenty-two animals were divided in 2 groups (A and B) and immunised with two doses of the vaccine (group A, n = 14) or used as unvaccinated control (group B, n = 8). Five months after the second vaccination, seven animals of group A and four animals of group B were challenged by nasal route with M. agalactiae. The remaining seven vaccinated and four control animals were challenged intranasally eight months after vaccination. The vaccine was able to induce a full-protective immunity preventing the clinical signs of contagious agalactia and the infection by M. agalactiae in all groups of animals irrespective of the time of challenge after booster administration. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Mycoplasma agalactiae is the major cause of contagious agalac- tia (CA), an infectious disease of small ruminants characterised by mastitis, agalactia, arthritis and keratoconjunctivitis. CA-like clini- cal signs either alone or associated with respiratory symptoms may be also caused by other mycoplasmas, including Mycoplasma capri- colum subsp. capricolum, Mycoplasma mycoides subsp. mycoides Large Colony, M. mycoides subsp. capri and Mycoplasma putrefaciens (DaMassa et al., 1992; Nicholas, 2002). Although in endemic areas CA is responsible for economic losses due to decreased milk pro- duction and to reduction of productive life of the infected animals, prophylactic measures are not always applied due to the lack of complete knowledge of the pathways of infection and to the lim- ited availability of highly effective vaccines. In the past, vaccines against CA were prepared using milk or or- gans from infected animals. Those vaccines were poorly effective and were able to transmit other pathogens such as the scrapie agent (Caramelli et al., 2001). Accordingly, in recent years several studies were made to develop novel safe and effective vaccines against M. agalactiae (Leon Vizcaino et al., 1995; Buonavoglia et al., 1998; Tola et al., 1999; Greco et al., 2002; de la Fe et al., 2007; Buonavoglia et al., 2008). Experimental vaccines combined with aluminium hydroxide [Al(OH) 3 ] were safe in sheep, but elic- ited low antibody titres that persisted no longer than three months (Buonavoglia et al., 1998; Tola et al., 1999; Greco et al., 2002). Con- versely, experimental mineral-oil adjuvanted vaccines were pro- ven to be highly immunogenic with high long-lasting (at least 11 months) antibody levels although the vaccine induced severe granulomatous reactions at the injection site (Buonavoglia et al., 1998; Greco et al., 2002). Recently, three vaccines against CA, pre- pared with different mineral-oil adjuvants, were tested in sheep, but the animals were challenged after only 21 days post-vaccina- tion which is in agreement with the European Pharmacopoeia, whereas no data were provided about the long-term duration of the elicited protection against M. agalactiae infection (Buonavoglia et al., 2008). In the present study, the long-term immunogenicity and pro- tection induced by an inactivated vaccine adjuvanted with Monta- nide ISA-563, Marcol-52 and Montane-80 in a 30:63:7 ratio were evaluated in sheep. 2. Materials and methods 2.1. Animals Twenty-two Comisana sheep, at the second lactation, were re- cruited for the study. The sheep were selected from a CA free flock. 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.07.006 * Corresponding author. Address: Department of Public Health and Animal Sciences, Faculty of Veterinary Medicine, University of Bari, Strada provinciale per Casamassima Km 3, 70010 Valenzano, Bari, Italy. Tel.: +390804679818; fax: +390804679843. E-mail address: d.buonavoglia@veterinaria.uniba.it (D. Buonavoglia). Research in Veterinary Science 88 (2010) 16–19 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc