Anaerobes in animal disease Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D Cl  ovis Moreira Jr. a, 1 , Carlos Eduardo Pouey da Cunha a, 1 , Gustavo Marçal Schmidt Garcia Moreira a, 2 , Marcelo Mendonça a , Felipe Masiero Salvarani b , ^ Angela Nunes Moreira a, c , Fabricio Rochedo Conceiç ~ ao a, * a Centro de Desenvolvimento Tecnol ogico, Núcleo de Biotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil b Faculdade de Medicina Veterin aria, Universidade Federal do Par a, Castanhal, PA, Brazil c Faculdade de Nutriç~ ao, Universidade Federal de Pelotas, RS, Brazil article info Article history: Received 14 January 2016 Received in revised form 15 April 2016 Accepted 24 May 2016 Available online 25 May 2016 Keywords: Clostridium botulinum Vaccine Recombinant bacterins Cell lysate vaccine Cattle botulism abstract Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 mg rH C C and rH C D each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with re- combinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry- friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Botulism is an intoxication caused by botulinum neurotoxins (BoNTs) produced mainly by Clostridium botulinum, a Gram- positive, ubiquitous, anaerobic, spore-forming, and rod-shaped bacterium that is also present in the gastrointestinal tract of ani- mals [1]. C. botulinum is classified into eight serotypes (A to H) according to their antigenicity [2,3]. BoNTs are comprised by a light chain (LC, 50 kDa) and a heavy chain (HC, 100 kDa), the latter composed of two 50 kDa domains. The translocation N-terminal domain (H N ) intermediates LC internalization, while the binding C- terminal domain (H C ) is responsible for interacting with cholinergic neuron receptors [4]. The metalloprotease activity of LC inside the neuron cells results in flaccid paralysis by impairing acetylcholine neuromuscular junctions [5,6]. Among these protein regions, H C is the main research target, since vaccines against this domain are able to induce antibodies that are capable of neutralizing intoxi- cation [7]. Cattle botulism is responsible for great financial losses, and has been one of the main causes of death in the last decades in Brazil [8e11]. BoNT serotypes C and D are responsible for causing cattle * Corresponding author. Laborat orio de Imunologia Aplicada, Centro de Desen- volvimento Tecnol ogico, Biotecnologia, UFPel, Brazil. E-mail address: fabricio.rochedo@ufpel.edu.br (F.R. Conceiç~ ao). 1 These authors contributed equally to this work. 2 Current address: Technische Universit€ at Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Spielmannstr. 7, 38106 Braunschweig, Germany. Contents lists available at ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe http://dx.doi.org/10.1016/j.anaerobe.2016.05.012 1075-9964/© 2016 Elsevier Ltd. All rights reserved. Anaerobe 40 (2016) 58e62