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Clostridium septicum (CS) is a pathogen that can cause the death of animals in livestock worldwide through its main virulence factor, alpha-toxin (ATX). The aspects involved in diseases caused by ATX, such as economic impact, prevalence, and rapid clinical course, require that animals should be systematically immunized. This review provides an overview of CS in livestock farming and discusses current immunization methods. Currently, commercial vaccines available against CS involve the cultivation and inactivation of microorganisms and toxins using a time-consuming, expensive, and high biological risk-carrying production platform, and some have been reported to be ineffective. An alternative to this process is the recombinant DNA technology, although recombinant ATX obtained thus far is no longer efficient in stimulating protective antibody titers despite improvements in the production methods. On the other hand, immunized animals have highly favorable levels of survival when subjected to challenge tests, suggesting that high titers of circulating serum antibodies may not be representative of protection after immunization and that the non-immune cellular defenses associated with the particularities of the mechanism of action of ATX may be involved in the immune response of the host. To contribute to the future of global livestock farming through the development of more efficient recombinant vaccines, we suggest novel perspectives and strategies, such as the location of immunodominant epitopes, expression of relevant functional domains, and construction of chimeras, in the rational design of recombinant ATX.

Phospholipases C (PLCs) represent an important group of lethal toxins produced by pathogenic bacteria of the Clostridium genus, including the beta toxin of C. haemolyticum. Bacillary hemoglobinuria in cattle and sheep is the main disease caused by this pathogen and its incidence can be reduced by annual vaccination of herds. Currently, widely used vaccines depend on cultivating the pathogen and obtaining high concentrations of the toxin, disadvantages that can be overcome with the use of recombinant vaccines. In the development of this new generation of immunizing agents, identifying and understanding the structural and immunological aspects of the antigen are crucial steps, but despite this, the beta toxin is poorly characterized. Fortunately, the time and resources required for these investigations can be reduced using immunoinformatics. To advance the development of recombinant vaccines, in addition to a brief review of the structural and immunological aspects of beta toxin, this work provides in silico mapping of immunodominant regions to guide future vaccinology studies against C. haemolyticum. A review of alternatives to overcome the limitations of beta toxin vaccines (conventional or recombinant) is also proposed.

The emergence of recombinant DNA technology has led to the exploration of the use of the technology to develop novel vaccines. With a fundamental role in vaccines design, several immunoinformatics tools have been created to identify isolated epitopes that stimulate a specific immune response, contributing to effective vaccines development. In the past, vaccine development projects relied entirely on animal experimentation, a relatively expensive and time-consuming process. Currently, use of immunoinformatics tools play a vital role in the antigen analysis and refinement, allowing the identification of possible protective epitopes capable of stimulating convenient humoral or cellular immune responses, in addition to facilitating time and cost reduction of vaccine production. The vaccination aimed at bacterial species of Clostridium spp. has been considered a promising example of use of these approaches in recent years. Based on the literature search, it is possible to understand the best immunoinformatics software used by researchers that facilitate recombinant vaccine antigens design and development. This chapter presents an overview of how these tools are supporting the antigen engineering, aiming at increasing the efficiency of inducing protective immune response in animals.

Farm animals are frequently affected by a group of diseases with a rapid clinical course, caused by Clostridium spp. and immunization is essential to provide protection. However, the current manufacturing platform for these vaccines has disadvantages and the main alternative is the use of an expression system that uses Escherichia coli to obtain recombinant vaccine antigens. In this chapter we describe procedures for cloning, expression and characterization of recombinant toxins from Clostridium spp. produced in E. coli for veterinary vaccine applications.

This chapter describes a practical, industry-friendly, and efficient vaccine protocol based on the use of Escherichia coli cell fractions (inclusion bodies or cell lysate supernatant) containing the recombinant antigen. This approach was characterized and evaluated in laboratory and farm animals by the seroneutralization assay in mice, thereby showing to be an excellent alternative to induce a protective immune response against clostridial diseases.

This chapter describes a practical, industry-friendly, and efficient vaccine protocol based on the use of Escherichia coli cell fractions (inclusion bodies or cell lysate supernatant) containing the recombinant antigen. This approach was characterized and evaluated in laboratory and farm animals by the seroneutralization assay in mice, thereby showing to be an excellent alternative to induce a protective immune response against clostridial diseases.

Farm animals are frequently affected by a group of diseases with a rapid clinical course, caused by Clostridium spp. and immunization is essential to provide protection. However, the current manufacturing platform for these vaccines has disadvantages and the main alternative is the use of an expression system that uses Escherichia coli to obtain recombinant vaccine antigens. In this chapter we describe procedures for cloning, expression and characterization of recombinant toxins from Clostridium spp. produced in E. coli for veterinary vaccine applications.

Clostridium perfringens type A is the causative agent of gas gangrene and gastroenteric (“yellow lamb disease”) disease in ruminants, with C. perfringens alpha toxin (CPA) being the main virulence factor in the pathogenesis of these illnesses. In the present study, we have developed recombinant Escherichia coli bacteria expressing rCPA and used it to vaccinate rabbits and sheep. Doses of up to 200 μg of rCPA used for inoculation, induced 13.82 IU.mL−1 of neutralizing antitoxin in rabbits, which is three times higher than that recommended by the USDA (4 IU.mL−1). In sheep, recombinant bacteria induced antitoxin titers of 4 IU.mL−1, 56 days after the first dose. rCPA which was expressed, mainly, in inclusion bodies, was not found to influence the immunogenicity of the vaccine. The recombinant Escherichia coli bacterin, produced simply and safely, is capable of affording protection against diseases caused by C. perfringens CPA. The current findings represent a novel production method for CPA vaccines potentially applicable to veterinary medicine.

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the β toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400μg) of C. perfringens α and β recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection. RESUMO: Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina β, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400μg) dos toxóides recombinantes α e β de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.

Beta toxins (CPB) produced by Clostridium perfringens type B and C cause various diseases in animals, and the use of toxoids is an important prophylactic measure against such diseases. Promising recombinant toxoids have been developed recently. However, both soluble and insoluble proteins expressed in Escherichia coli can interfere with the production and immunogenicity of these antigens. In this context, bioinformatics tools have been used to design new versions of the beta toxin, and levels of expression and solubility were evaluated in different strains of E. coli. The immunogenicity in sheep was assessed using the molecule with the greatest potential that was selected on analyzing these results. In silico analyzes, greater mRNA stability (−169.70 kcal/mol), solubility (−0.755), and better tertiary structure (−0.12) were shown by rCPB-C. None of the strains of E. coli expressed rFH8-CPB, but a high level of expression and solubility was shown by rCPB-C. Higher levels of total and neutralizing anti-CPB antibodies were observed in sheep inoculated with bacterins containing rCPB-C. Thus, this study suggests that due to higher productivity of rCPB-C in E. coli and immunogenicity, it is considered as the most promising molecule for the production of a recombinant vaccine against diseases caused by the beta toxin produced by C. perfringens type B and C.

Many studies have suggested that imbalance of the gut microbial composition leads to an increase in pro-inflammatory cytokines and promotes oxidative stress, and this are directly associated with neuropsychiatric disorders, including major depressive disorder (MDD). Clinical data indicated that the probiotics have positive impacts on the central nervous system and thus may have a key role to treatment of MDD. This study examined the benefits of administration of Komagataella pastoris KM71H (8 log UFC·g–1/animal, intragastric route) in attenuating behavioral, neurochemical, and neuroendocrine changes in animal models of depressive-like behavior induced by repeated restraint stress and lipopolysaccharide (0.83 mg/kg). We demonstrated that pretreatment of mice with this yeast prevented depression-like behavior induced by stress and an inflammatory challenge in mice. We believe that this effect is due to modulation of the permeability of the blood-brain barrier, restoration in the mRNA levels of the Nuclear factor kappa B, Interleukin 1β, Interferon γ, and Indoleamine 2 3-dioxygenase, and prevention of oxidative stress in the prefrontal cortices, hippocampi, and intestine of mice and of the decrease the plasma corticosterone levels. Thus, we conclude that K. pastoris KM71H has properties for a new proposal of probiotic with antidepressant-like effect, arising as a promising therapeutic strategy for MDD.

Botulism is a paralytic disease caused by the intoxication of neurotoxins produced by Clostridium botulinum. Among the seven immunologically distinct serotypes of neurotoxins (BoNTs A − G), serotypes C and D, or a chimeric fusion termed C/D or D/C, are responsible for animal botulism. The most effective way to prevent botulism in cattle is through vaccination; however, the commercially available vaccines produced by detoxification of native neurotoxins are time-consuming and hazardous. To overcome these drawbacks, a non-toxic recombinant vaccine was developed as an alternative. In this study, the recombinant protein vaccine was produced using an Escherichia coli cell-based system. The formaldehyde-inactivated E. coli is able to induce 7.45 ± 1.77 and 6.6 ± 1.28 IU/mL neutralizing mean titers against BoNTs C and D in cattle, respectively, determined by mouse neutralization bioassay, and was deemed protective by the Brazilian legislation. Moreover, when the levels of anti-BoNT/C and D were compared with those achieved by the recombinant purified vaccines, no significant statistical difference was observed. Cattle vaccinated with the commercial vaccine developed 1.33 and 3.33 IU/mL neutralizing mean titers against BoNT serotypes C and D, respectively. To the best of our knowledge, this study is the first report on recombinant E. coli bacterin vaccine against botulism. The vaccine was safe and effective in generating protective antibodies and, thus, represents an industry-friendly alternative for the prevention of cattle botulism.