Vaccination of cattle with a recombinant bivalent toxoid against botulism serotypes C and D

Abstract

Cattle botulism is a fatal intoxication caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D resulting in economic losses. Vaccination is the most effective way to control botulism. However, the commercially available vaccines are difficult and hazardous to produce. Neutralizing antibodies against the C-terminal fragment of the BoNT heavy chain (H_C) are known to protect against lethal doses of BoNTs. We report the vaccination of cattle with a previously tested recombinant chimera consisting of Escherichia coli heat-labile enterotoxin B subunit and the H_C of BoNTs C and D. Vaccinated animals produced neutralizing antibodies against serotypes C and D averaging 5 ± 0 and 6.14 ± 1.06 IU/mL, respectively. For BoNT D, the titers were greater than those measured for the commercial vaccine, which induced titers of 5 ± 0 and 2.85 ± 1.35 against the respective serotypes, suggesting that this chimera is effective against cattle botulism.

Introduction

Clostridium botulinum is a Gram-positive, anaerobic, spore-forming bacillus that is ubiquitous in nature and capable of growing in decaying organic matter. Under anaerobic conditions, it produces botulinum neurotoxins (BoNTs), which are responsible for causing botulism, a fatal intoxication characterized by flaccid paralysis due the inhibition of acetylcholine release at the neuromuscular junction [1]. These toxins are classified into seven serotypes (A to G) according to their antigenic differences, although they present similar pharmacological mechanisms [2]. BoNT serotypes C and D are the most common toxins for outbreaks of cattle botulism in many countries, including Brazil [3], [4], [5], [6], where it frequently presents fatality rates up to 100% [7]. Thus, bovine botulism is considered a cause of great economic loss.

The induction of neutralizing antibodies through vaccination is the best approach to control botulism [8]. Currently, vaccines against botulism are produced with formaldehyde-inactivated neurotoxins (toxoids) mixed with aluminum hydroxide as an adjuvant. Although efficient, this methodology presents some drawbacks: (1) the amount of native BoNT production in vitro is unpredictable and typically low; and (2) BoNTs are the most potent toxins known to mankind [9], [10], requiring high levels of biosafety. Therefore, new approaches, such as the development of recombinant vaccines, appear to be promising to overcome these problems.

A recombinant chimera comprising LTB, a potent adjuvant of the humoral immune response, and the C-terminal fragments of BoNT serotypes C and D was previously produced and tested [11]. This construct was able to induce high levels of neutralizing antibodies (5 and 10 International Units per milliliter (IU/mL) against BoNTs C and D, respectively) in guinea pigs when administered with aluminum hydroxide as an adjuvant. The objective of the present work was to evaluate whether our construct was capable of inducing a protective immune response in cattle.