Clostridium (C.) perfringens is the causative agent of several diseases and enteric infections in animals and humans. The pathogenicity of the bacterium is largely mediated by the production of a wide range of toxins. Individual C. perfringens strains produce only subsets of this toxin repertoire, which permits the classification in seven toxinotypes (A–G). In addition, a variety of minor toxins further characterizes the single strains. The aim of this work was to evaluate, using Polymerase Chain Reaction (PCR) assays, the diversity of 632 C. perfringens strains isolated in Italy over 15 years. The genotyped strains were analyzed to determine the presence of major and minor toxins (cpe, consensus, and atypical cpb2), their geographical origins, and the source of isolation (animal species or food). Our study shows that toxinotype A had the greatest representation (93%) and correlated mainly with consensus cpb2 in a variety of animal species, as well as with atypical cpb2 in the five food samples. Type D, associated with cpe and atypical cpb2 minor toxins, was identified in 3% of the cases, and type F was identified in 2.5%. Seven type C isolates (1.1%) were detected in cattle, whereas the only type B atypical cpb2 isolated in Italy was detected in a goat, and one type E cpe+atypical cpb2 was detected in a sheep. Type G was not detected.

Background: African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The African Swine Fever Virus (ASFV) has a genome characterized by a very complex DNA (170-193 kb) that encodes for more than 200 different proteins. Among these, the highly immunogenic phosphoprotein p30 plays a fundamental role in the induction of specific antibodies. To date, the lack of a vaccine against the disease requires continuous studies to improve knowledge about the virus and the development of new tests in addition to virological ones. Aim: The aim of this work was to produce specific monoclonal antibodies (mAbs) against the p30 protein of ASFV, which could find useful applications in routine diagnostics and the implementation of new diagnostic tools. Methods: ASFV p30 encoding gene was amplified and used for the generation of the recombinant baculovirus by transfection of the Sf21 insect cells. The recombinant protein was analyzed by immunofluorescence assay, purified, and used for mice Balb-c immunization. The hybridomas obtained were cultured and screened, using an indirect Enzyme-linked Immunosorbent Assay (iELISA), in order to select clones that secrete the mAbs of interest. Results: The expression of recombinant p30 protein was assessed using direct Immunofluorescence. The purified p30 protein fractions were analyzed by Coomassie gels staining confirming the presence of bands with a molecular weight of 30 kDa and used for the immunization of Balb-c mice. Six clones of pure hybridomas secreting the specific mAbs against recombinant p30 were obtained and tested in iELISA. The mAbs were also characterized by Western blot and Immunofluorescence assay. The best results were obtained with the anti-p30 mAb 2B8E10 clone which showed high reactivity with both recombinant and viral p30 protein, respectively. Conclusion: In this work, a recombinant p30 protein produced in an insect cell system was purified and used to immunize Balb-c mice. Six anti-p30 mAbs-secreting hybridomas clone cells were obtained. These mAbs displayed high reactivity against the recombinant protein, but only 2B8E10 mAb showed excellent functionality against the p30 protein produced by ASFV. These results open the possibility to develop different diagnostic assays.

Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin. In this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2Δ1–25-His6) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2Δ1–25-His6 was obtained after purification by Ni2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2Δ1–25-His6. Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins. The high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.