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The aim of this study was to evaluate the titers of neutralizing antibodies in cattle inoculated with multivalent commercial clostridial vaccines containing C. botulinum type C (BoNTC), C. botulinum type D (BoNTD), and C. perfringens epsilon (ETX) toxoids for a period of one year. Cattle (Bos taurus), aged 4-6 months and not previously immunized, were vaccinated under four different protocols at days 0 and 30 and followed over one year. Individual serum titration was performed by a serum neutralization test in mice or in MDCK cells. The number of animals with detectable neutralizing antibodies ranged from 40.6% to 78.1%, but only 12.5% of animals showed neutralizing antibodies against all tested antigens. Neutralizing antibodies were found only until 60 days for ETX, 120 days for BoNTC, and 180 days for BoNTD. The absence of detectable neutralizing antibodies against the three antigens before 360 days, suggests that cattle remained unprotected for a long period before the recommended booster vaccination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019. Published by Elsevier Ltd.)

Background: It is about 4 decades from the identification of Enterohemorrhagic Escherichia coli (EHEC) as a food-borne pathogen. There are many shreds of evidence that the bacteria are significant sources of intestinal infections and outbreaks even in developed countries. Developing an effective vaccine against O157 and non-O157 serotypes of EHEC is a good strategy to combat the bacteria. Raising antibody against main toxicity, adhering and colonizing apparatus of the bacteria using a multi-antigenic protein can be hopefully applicable., Material and Methods: A synthetic cassette consists of HcpA-EspA-Tir-Stx2B (HETS) subunit proteins were constructed and sub-cloned in pET32a (+). The protein was expressed and purified with Ni-NTA column and the BALB/c mice were immunized by the purified protein. HETS protein efficacy to elicit immune responses, O157 fecal shedding and immunity against Stx toxin were assessed. In addition, the cellular assays were performed to investigate the immune sera capability for neutralizing of Stx toxin and bacterial attachment apparatus., Results: The HETS protein (611 amino acid length) was expressed and validated by Western blotting. Exerted EHEC bacteria ratio in the immunized mice was reduced close to 60% in shedding test. Cellular assays revealed that the sera of the immunized animals were able to neutralize Stx holotoxin in an extent of 70%; also, immunized mice were able to tolerate up to 200 LD 50 of the active toxin. Moreover, toxin neutralization assay showed the capability of the immunized sera to block the cell adhesion., Conclusion: Regarding a lack of an efficient vaccine against EHEC, the proposed candidate immunogen, which consists of main adhesion and invasion factors, can overcome the lack of a vaccine against the bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The epsilon toxin (Etx) produced by Clostridium perfringens type B and D causes severe enterotoxaemia associated with a general edema and neurological alterations, leading to subsequent death and is listed as one of the most lethal toxins. Currently employed vaccines against C. perfringens epsilon toxin include toxoid based vaccines. Use of peptide vaccines has become an interesting approach for vaccination after the successful licensing of peptide vaccines against Haemophilus influenza, Neisseria meningitides and Streptococcus pneumonia that have demonstrated the potential and effectiveness of these vaccines. Therefore, the present study was undertaken to develop a peptide based vaccine against epsilon toxin. Peptides were selected on the basis of epitope mapping by making 35 overlapping peptides of 15 amino acid residues in length specific to the primary amino acid sequence of the toxin, with a 7 amino acid residues overlaps between sequential peptides. Chemically synthesized peptides that were recognised by the antibody against the full length epsilon toxin were further assessed for vaccine potential. The selected peptides were chemically conjugated to partially reduced tetanus toxoid (TT) using of N-succinimidyl-3(2-pyridyldithio) propionate. Immunization of BALB/c mice with TT-peptide conjugates by sub-cutaneous route induced sustained high level mixed immune response as analyzed by antibody isotyping. Immunoblot analysis and ELISA clearly indicated generation of Etx-specific antibodies. Further, neutralization studies with the antisera generated against the TT-conjugated peptide(s) demonstrated that the antisera were able to neutralize the lethal dose of epsilon toxin in vitro demonstrating its potential as a promising vaccine candidate against enterotoxaemia., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 μg rHCC and rHCD 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 recombinant 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the present study the efficacy Botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of Clostridium botulinum types C and D) was evaluated in four Danish dairy cows under field conditions. Other four dairy herds were unvaccinated. Blood serum of all animals was analyzed for specific C. botulinum types A, B, C, D and E antibodies using a developed ELISA. Feces of all animals were analyzed for botulinum neurotoxins (BoNTs) and C. botulinum spores. C. botulinum types C and D antibodies were significantly (p < 0.05) increased in vaccinated animals. Vaccination with botulism vaccine significantly reduced (p < 0.001) BoNTs and C. botulinum spores in cattle feces. Our findings represent that C. botulinum vaccination increases specific blood serum antibodies and reduces free BoNTs and C. botulinum spores in feces., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The level of circulating tetanus toxin, antitoxin and their individual influence on the outcome of tetanus cases were determined in unimmunized 125 neonatal and 39 infant cases of tetanus. PHA (passive haemagglutination) test showed 40% positive cases for toxin while its absence in the remaining cases indicated of either toxin fixation to the central nervous system (CNS) or it got neutralized by antitoxin. TN (toxin neutralization) and PHA test carried out in 46 sera samples revealed a strong positive correlation (r = 0.9) showing that 35/46 (76%) and 38/46 (82.6%) samples were positive for antitoxin, respectively. 25.4% of the neonate and infant cases and 34% of the control group had a protective serum tetanus antitoxin level. 42.5% of the paired sera from unimmunized mothers and their neonates showing nonprotective antitoxin levels suggested that a high level of antitoxin is needed for transplacental transfer, although transfer may not play a decisive role in the resistance against the disease. The presence of toxin or antitoxin in the clinical cases did not affect the outcome of the disease, although in neonates, presence of toxin was found to be a bad prognostic sign. This study explicitly advocates for the need to improve the vaccination coverage strategy., (Copyright © 2012 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.

To define the alterations provoked by malnutrition during suckling (20 pups/dam) in the gut-associated lymphoid tissues of rats, Peyer patch (PP) and mesenteric lymph node (MLN) cells were studied by flow cytometry. After weaning (21 days of age), rats malnourished during suckling (MNR) showed an increase in the CD4+ CD45RC+ subset together with a decrease in the CD4+ CD45RC- subset (P < 0.01). These alterations remained even after 3 weeks of refeeding with stock diet. The CD4+CD8+ subset was not increased in the MNR, indicating that a release of cortical thymocytes did not occur. At weaning the percentage of CD4+Thy1+ cells was decreased in the MNR, indicating a low number of cells released from the thymus. When the B cell lineage was studied, we found a decreased percentage of precursors in the bone marrow and a decreased percentage of mature B cells in the periphery. When the MNR were immunized intra-PP with cholera toxin (CT) after 1 week of refeeding, the specific IgG and IgA and IgM antibody-forming cells (measured by ELISPOT) were diminished in the PP, MLN, and spleen when compared to the age-matched controls (P < 0.001). These results were coincident with the ELISA titers obtained in the sera and in the intestinal fluids. When CT was administered after 2 weeks of refeeding, the number of IgM anti-toxin AFC approached control values, but the number of IgA and IgG AFC continued to be low. When 3 weeks of refeeding was allowed before the CT delivery, the immune response in the MNR approached control values. These results indicate that malnutrition during suckling provokes alterations in B and T lymphocytes and produces a lack in the induction of the primary and secondary immune responses in the GALT which reversed after 3 weeks of refeeding.