An in vitro assay for toxicity testing of Clostridium perfringens type C ß-toxin.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the ß-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the ß-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for ß-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the ß-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

Multivalent poultry vaccine development using Protein Glycan Coupling Technology.

BACKGROUND: Poultry is the world's most popular animal-based food and global production has tripled in the past 20 years alone. Low-cost vaccines that can be combined to protect poultry against multiple infections are a current global imperative. Glycoconjugate vaccines, which consist of an immunogenic protein covalently coupled to glycan antigens of the targeted pathogen, have a proven track record in human vaccinology, but have yet to be used for livestock due to prohibitively high manufacturing costs. To overcome this, we use Protein Glycan Coupling Technology (PGCT), which enables the production of glycoconjugates in bacterial cells at considerably reduced costs, to generate a candidate glycan-based live vaccine intended to simultaneously protect against Campylobacter jejuni, avian pathogenic Escherichia coli (APEC) and Clostridium perfringens. Campylobacter is the most common cause of food poisoning, whereas colibacillosis and necrotic enteritis are widespread and devastating infectious diseases in poultry. RESULTS: We demonstrate the functional transfer of C. jejuni protein glycosylation (pgl) locus into the genome of APEC χ7122 serotype O78:H9. The integration caused mild attenuation of the χ7122 strain following oral inoculation of chickens without impairing its ability to colonise the respiratory tract. We exploit the χ7122 pgl integrant as bacterial vectors delivering a glycoprotein decorated with the C. jejuni heptasaccharide glycan antigen. To this end we engineered χ7122 pgl to express glycosylated NetB toxoid from C. perfringens and tested its ability to reduce caecal colonisation of chickens by C. jejuni and protect against intra-air sac challenge with the homologous APEC strain. CONCLUSIONS: We generated a candidate glycan-based multivalent live vaccine with the potential to induce protection against key avian and zoonotic pathogens (C. jejuni, APEC, C. perfringens). The live vaccine failed to significantly reduce Campylobacter colonisation under the conditions tested but was protective against homologous APEC challenge. Nevertheless, we present a strategy towards the production of low-cost "live-attenuated multivalent vaccine factories" with the ability to express glycoconjugates in poultry.

Cattle and goats' humoral response to vaccination with Clostridium perfringens type D purified epsilon toxoids.

Herd vaccination is an important preventive measure against enterotoxemia in ruminants. Vaccination in goats should be performed every four months, and recent studies have shown that immunity in cattle lasts for less than one year. One of the mechanisms for increasing the duration of the immune response is to use purified toxoids as immunogens. The aim of the present study was to evaluate the humoral response in cattle and goats after vaccination with purified and semi-purified Clostridium perfringens type D epsilon toxoid. The following three different vaccines were used: vaccine 1 (V1), a semi-purified toxoid adsorbed to aluminum hydroxide; vaccine 2 (V2), a purified toxoid adsorbed to aluminum hydroxide; and vaccine (V3), a purified toxoid adsorbed on chitosan microparticles. Groups of cattle (n = 6-7) and goats (n = 6-7) were vaccinated on days 0 and 30, and serum samples for antitoxin titration were collected every 30 days for one-year post-vaccination. Goats were revaccinated on day 360, and their serum was evaluated on days 367 and 374. The antibody peaks ranged between 6.90 and 11.47 IU/mL in cattle and from 1.11 to 4.40 IU/mL in goats. In cattle administered with the V1 and V2 vaccines, we observed that the antibody titers were maintained above 0.2 IU/mL until the end of the experiment. In goats, V2 elicited long-lasting antibodies, and all animals maintained the protective titers for 210 days after the first dose. In conclusion, the purified toxoid vaccine with aluminum hydroxide adjuvant was able to induce strong and long-lasting humoral responses in both species and could be an alternative for improving the immunization schedule against enterotoxemia in goats and cattle.

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha, Beta and Epsilon of Clostridium perfringens.

The alpha (CPA), beta (CPB) and epsilon (ETX) toxins of Clostridium perfringens are responsible for causing diseases that are difficult to eradicate and have lethal potential in production animals. Vaccination of herds is still the best control strategy. Recombinant clostridial vaccines have shown good success at inducing neutralizing antibody titers and appear to be a viable alternative to the conventional production of commercial clostridial toxoids. Research is still needed on the longevity of the humoral immune response induced by recombinant proteins in immunized animals, preferably in target species. The objective of this study was to measure the humoral immune response of cattle immunized with trivalent vaccines containing the recombinant proteins alpha (rCPA), beta (rCPB) and epsilon (rETX) of C. perfringens produced in Escherichia coli at three different concentrations (100, 200, and 400 µg) of each protein for 12 months. The recombinant vaccines containing 200 (RV2) and 400 µg (RV3) yielded statistically similar results at 56 days. They performed better throughout the study period because they induced higher neutralizing antibody titers and were detectable for up to 150 and 180 days, respectively. Regarding industrial-scale production, RV2 would be the most economical and viable formulation as it achieved results similar to RV3 at half the concentration of recombinant proteins in its formulation. However, none of the vaccines tested induced the production of detectable antibody titers on day 365 of the experiment, the time of revaccination typically recommended in vaccination protocols. Thus, reiterating the need for research in the field of vaccinology to achieve greater longevity of the humoral immune response against these clostridial toxins in animals, in addition to the need to discuss the vaccine schedules and protocols adopted in cattle production.

Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months.

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry's production of vaccines against the action of C. perfringens in equine species.

Immunostimulatory Potential of Fruits and Their Extracts in Poultry.

The impact of antibiotic use for growth promotion in livestock and poultry production on the rise of antimicrobial resistance (AMR) in bacteria led to the ban of this practice in the European Union in 2006 and a restriction of antimicrobial use (AMU) in animal agriculture in Canada and the United States of America. There is a high risk of infectious diseases such as necrotic enteritis due to Clostridium perfringens, and colibacillosis due to avian pathogenic Escherichia coli in antimicrobial-free broiler chickens. Thus, efficient and cost-effective methods for reducing AMU, maintaining good poultry health and reducing public health risks (food safety) are urgently needed for poultry production. Several alternative agents, including plant-derived polyphenolic compounds, have been investigated for their potential to prevent and control diseases through increasing poultry immunity. Many studies in humans reported that plant flavonoids could modulate the immune system by decreasing production of pro-inflammatory cytokines, T-cell activation, and proliferation. Fruits, especially berries, are excellent sources of flavonoids while being rich in nutrients and other functionally important molecules (vitamins and minerals). Thus, fruit byproducts or wastes could be important resources for value-added applications in poultry production. In the context of the circular economy and waste reduction, this review summarizes observed effects of fruit wastes/extracts on the general health and the immunity of poultry.

Evaluation of different antigenic preparations against necrotic enteritis in broiler birds using a novel Clostridium perfringens type G strain.

OBJECTIVE: Keeping in view, the constraints faced by the Indian broiler industry with lack of a suitable vaccine against Necrotic Enteritis (NE), a study has been proposed to explore the prevalence and detail characterization of C. perfringens type G in NE suspected broiler chicken in the process of suitable vaccine development. METHODS: Intestinal scrapings/faecal contents of NE suspected broiler chickens were screened to establish the prevalence of C.perfringens type G in broiler birds. A most pathogenic, highly resistant type G isolate of C. perfringens, bearing both tpeL and gapC gene was selected for preparation of three different vaccine formulations, and to evaluate their immunogenic potential in broiler birds. RESULTS: Screening of clinical samples of NE suspected broiler birds revealed C. perfringens type G, bearing gapC gene in 51.22% samples, of which 47.62% revealed tpeL gene. Seven of the tpeLpos type G isolates were comparatively more pathogenic for mice, of which, one exhibited multidrug resistance towards ciprofloxacin, norfloxacin, tetracycline and levofloxacin. The sonicated supernatant (SS) prepared from the selected tpeL and gapC positive isolate could maintain a significantly higher protective IgG response than toxoid and bacterin preparation from the 21st to 28thday of age in immunized birds. CONCLUSION: The additional TpeL toxin in C. perfringens type G has been proved to be an additional key biological factor in the pathogenesis of NE in broiler chickens. Considering the release of more immunogenic proteins, the SS proved to be a better immunogenic preparation against NE with a multiple immunization dose.

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies.

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE ADULT SPRINGBOK (ANTIDORCAS MARSUPIALIS), IMPALA (AEPYCEROS MELAMPUS), ALPACA (VICUGNA PACOS), AND RED-NECKED WALLABY (MACROPUS RUFOGRISEUS) OVER A YEAR.

Enterotoxemia is an important issue in various zoological taxa. In this study, serologic responses over a 1-yr period after vaccination with a multivalent clostridial vaccine were evaluated in 10 adult springboks (Antidorcas marsupialis), 12 impalas (Aepyceros melampus), seven alpacas (Vicugna pacos), and five red-necked wallabies (Macropus rufogriseus). Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay and determined as the percentage of inhibition (% inhib). Initial % inhib was (0.01-18.9)%. All animals received initial vaccination with a booster vaccine 4 weeks apart. Serum samples were collected at T0 (nonvaccinated), 15, 30, 60, 180, and 360 days postvaccination (dpv) for analysis. The vaccine induced a high antibody response that peaked at 15, 30, and 60 dpv in springboks, 30 and 60 dpv in impalas (P < 0.01), and 60 dpv in alpacas and wallabies (P < 0.01). The booster vaccine was followed by a high antibody response, which slowly decreased with time. The antibody response was significantly higher at 360 dpv than at T0 in wallabies and alpacas (P < 0.01). In impalas and springboks, it appeared that a booster every 6 mo might be required to maintain an antibody response above baseline (P < 0.01). Because no challenge studies were performed, it is unknown whether the measured humoral immune responses would have been protective. Further research is warranted to investigate protective effects of antibodies to inoculation challenge in nondomestic species.

Characterization of immunological properties of chicken chemokine CC motif ligand 5 using new monoclonal antibodies.

CCL5 (formerly RANTES) belongs to the CC (or ß) chemokine family and is associated with a plethora of inflammatory disorders and pathologic states. CCL5 is mainly produced and secreted by T cells, macrophages, epithelial cells, and fibroblasts and acts as a chemoattractant to recruit effector cells to the inflammation sites. Chicken CCL5 (chCCL5) protein is closely related to avian CCL5 orthologs but distinct from mammalian orthologs, and its modulatory roles in the immune response are largely unknown. The present work was undertaken to characterize the immunological properties of chCCL5 using the new sets of anti-chCCL5 mouse monoclonal antibodies (mAbs). Eight different mAbs (6E11, 6H1, 8H11, 11G1, 11G11, 12H1, 13D1, and 13G3) were characterized for their specificity and binding ability toward chCCL5. Two (13G3 and 6E11) of them were selected to detect native chCCL5 in chCCL5-specific antigen-capture ELISA. Using 13G3 and 6E11 as capture and detection antibodies, respectively, the ELISA system detected serum chCCL5 secretions in Clostridium perfringens- and Eimeria-infected chickens. The intracellular expressions of chCCL5 in primary cells or cell lines derived from chickens were validated in immunocytochemistry and flow cytometry assays using both 13G3 and 6E11 mAbs. Furthermore, 6E11, but not 13G3, neutralized chCCL5-induced chemotaxis in vitro using chicken PBMCs. These molecular characteristics of chCCL5 demonstrate the potential application of anti-chCCL5 mAbs and CCL5-specific antigen-capture detection ELISA for detecting native chCCL5 in biological samples. The availability of these new immunological tools will be valuable for fundamental and applied studies in avian species.

Effects of heat-killed Enterococcus faecalis T-110 supplementation on gut immunity, gut flora, and intestinal infection in naturally aged hamsters.

Infectious diseases are a threat to elderly individuals, whose immune systems weaken with age. Among the various infectious diseases, Clostridium difficile infection is associated with a high rate of mortality in elderly individuals and is a serious health problem worldwide, owing to the increasing infection rates. Probiotic use has been proposed as an effective countermeasure for C. difficile infection. The aim of this study was to evaluate the effects of heat-killed Enterococcus faecalis T-110 on intestinal immunity, intestinal flora, and intestinal infections, especially C. difficile infections, in naturally ageing animals, for extrapolating the results to elderly human subjects. Twenty female hamsters were randomly distributed into two groups. Group 1 was fed a basal diet and group 2 was fed a basal diet supplemented with heat-killed E. faecalis for 7 days. Heat-killed E. faecalis T-110 improved the gut immunity and microflora, especially Clostridium perfringens and C. difficile, in naturally aged hamsters. Therefore, heat-killed E. faecalis T-110 use may be a countermeasure against age-related immune dysfunction and intestinal infections, especially C. difficile infection, in elderly humans. However, further investigation in this regard is needed in humans.

Effectiveness of Chitosan Nanoparticles in Development of Pentavalent Clostridial Toxoid Vaccine in Terms of Clinical Pathology Elements and Immunological Responses.

Toxoid vaccines can provide protective immunity against clostridial diseases. Since the duration of the toxoid vaccine immunogenicity is short, these vaccines need to contain an adjuvant. The nanoparticles of chitosan can stimulate humoral and cell-mediated immune responses. In the present study, the effect of chitosan nanoparticles was investigated on the immunogenicity of the pentavalent clostridial toxoid vaccine containing Clostridium perfringens types D, C, and B, Clostridium septicum, as well as Clostridium novyi. Rabbits were immunized by two injections with 3-week intervals and checked clinically and through autopsy 2 weeks after the last injection. Hematological changes were investigated during immunization, including the changes of white and red blood cell counts, hemoglobin, packed cell volume, platelet, neutrophil, lymphocyte, eosinophil, basophile, monocyte, and Neut/Lymph. Biochemical factors, namely creatinine, blood urea nitrogen, glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, and albumin, were also studied. The changes in immune responses during the immunization period were investigated through indirect enzyme-linked immunosorbent assay (ELISA). The results of ELISA showed that chitosan significantly enhanced immunogenicity when accompanied with in the pentavalent clostridial toxoid vaccine. During the immunogenicity period and following that, no changes were observed in clinical behavior and internal organs after autopsy. The hematological and biochemical factors were reported with no significant pathologic changes during immunization in the control and vaccinated groups (p &lt;0.05). The obtained findings revealed that the toxoid vaccines could not induce significant physiological changes in the body. The vaccine containing chitosan could stimulate humoral immunity 2-3 times higher than the nonchitosan vaccine. The humoral immune response was significantly duplicated due to the chitosan effect. Chitosan not only had no local or general side effects but also could be a good help with the enhancement of the immune system; therefore, it can be recommended as an appropriate safe adjuvant in the development of toxoid vaccines.

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

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 ß 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 ß 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.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, 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 ß 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.(AU)

Clostridium perfringens phospholipase C impairs innate immune response by inducing integrated stress response and mitochondrial-induced epigenetic modifications.

Clostridium perfringens, a rod-shaped, gram-positive, anaerobic, spore-forming bacterium is one of the most widely occurring bacterial pathogens, associated with a spectrum of diseases in humans. A major virulence factor during its infection is the enzyme phospholipase C encoded by the plc gene, known as Clostridium perfringens phospholipase C (CpPLC). The present study was designed to understand the role of CpPLC in inducing survival mechanisms and mitochondrial-induced epigenetic changes in a human lymphocyte cell culture model. Following exposure to CpPLC, a significant generation of mitochondrial reactive oxygen species was observed, which coincided with the changes in the expression of vital components of MAP/ERK/RTK signaling cascade that regulates the downstream cellular functions. These disturbances further led to alterations in the mitochondrial genome and functioning. This was supported by the observed upregulation in the expression of mitochondrial fission genes Drp1, Fis1, and Mff, and mitochondrial fusion genes MFN1, MFN2, and OPA1 following CpPLC exposure. CpPLC exposed cells showed upregulation of OMA1, DELE1, and HRI genes involved in the integrated stress response (ISR), which suggests that it may induce the ISR that provides a pro-survival mechanism to the host cell. CpPLC also initiated immune patho-physiologic mechanisms including mitochondrial-induced epigenetic modifications through a mitochondrial-ROS driven signaling pathway. Interestingly, epigenetic machinery not only play a pivotal role in lymphocyte homeostasis by contributing to cell-fate decisions but thought to be one of the mechanisms by which intracellular pathogens survive within the host cells. Importantly, the impairment of mtDNA repair among the CpPLC exposed cells, induced alterations within mtDNA methylation, and led to the deregulation of MT-CO1, MT-ND6, MT-ATPase 6, and MT-ATPase8 gene expression profiles that are important for mitochondrial bioenergetics and subsequent metabolic pathways. This was further confirmed by the changes in the activity of mitochondrial electron chain complexes (complex I, II, III, IV and V). The altered mtDNA methylation profile was also found to be closely associated with the varied expression of mitomiRs and their targets. CpPLC exposed cells showed up-regulation of miR24 expression and down-regulation of miR34a, miR150, and miR155, while the increased expression of mitomiR target genes i.e. of K-Ras, MYC, EGFR, and NF-kß was also observed in these cells. Altogether, our findings provide novel insights into the derailment of redox signaling machinery in CpPLC treated lymphocytes and its role in the induction of survival mechanisms and mitochondrial-induced epigenetic modifications.

Bioinformatics analysis of NetF proteins for designing a multi-epitope vaccine against Clostridium perfringens infection.

Clostridium perfringens is an important human and animal pathogen that is the primary causative agent of necrotizing enteritis and enterotoxemia in many types of animals. C. perfringens produces a variety of toxins, including NetF which may plays a crucial role in the pathogenesis of foal and canine necrotizing enteritis. In this study, we used several bioinformatics methods to analyze various aspects of the NetF proteins, including the physicochemical properties, secondary and tertiary structures, and the dominant B-cell and T-cell epitopes. The results showed that NetF protein was a stable and hydrophilic protein. The secondary structure of the NetF protein consisted of 2.62% alpha helixes, 6.56% beta turns, 38.69% extended strands and 52.13% random coils. Moreover, several potential B and T-cell epitopes were identified for NetF. In addition, the obtained findings from antigenicity and allergenicity evaluation remarked that this protein is immunogenic and non-allergen. Based on the results of Ramachandran plot, 94.22%, 5. 42%, and 0.36% of amino acid residues were incorporated in the favored, allowed, and outlier regions, respectively. This study provides a foundation for further investigations, and laid a theoretical basis for the development of an appropriate vaccine against C. perfringens infection.

An atypical lipoteichoic acid from Clostridium perfringens elicits a broadly cross-reactive and protective immune response.

Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(ß1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.

Immunization of mice against alpha, beta, and epsilon toxins of Clostridium perfringens using recombinant rCpa-b-x expressed by Bacillus subtilis.

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.

Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies.

BACKGROUND: Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the C-terminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms. RESULTS: In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC. CONCLUSIONS: The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

In silico design and in vitro analysis of a recombinant trivalent fusion protein candidate vaccine targeting virulence factor of Clostridium perfringens.

Necrotic enteritis (NE) is a multifactorial disease in broiler that is caused by colonization of Clostridium perfringens in their gastrointestinal tract. Recently several immunogenic proteins from virulent C. perfringens have been considered as vaccines to provide protection against NE. In this study, a novel trivalent fusion protein including immunogenic epitopes of three virulence factors of, NetB, alpha toxin and a metallopeptidase protein (NAM) was designed using in silico studies. Circular dichroism spectra was applied for determination of secondary structure and folding properties of the purified recombinant NAM (rNAM) expressed in E. coli. The antigenicity of rNAM was confirmed by induction of immune response in rabbit and neutralization experiments of the toxins in cell culture studies. To this end, anti-rNAM antisera neutralized the crude toxins produced by a wild type virulent C. perfringens strain using chicken hepatocellular carcinoma (LMH) cell lines. The cells were exposed to a mixture of anti-rNAM antisera and 2 × LD50 doses of the toxins. The result showed 94% viability of the cells against the crude toxins, in the presence of anti-rNAM antisera. Our study suggests that combination of metallopeptidase protein along with alpha toxin and NetB toxins is a potent immunogen which is able to neutralize the toxicity of crude extracellular toxins. The recombinant chimeric NAM could be a suitable and effective subunit vaccine candidate to prevent NE disease caused by C. perfringens.

Protective Effects of Lactobacillus plantarum 16 and Paenibacillus polymyxa 10 Against Clostridium perfringens Infection in Broilers.

This study aimed to investigate the protective effects of Lactobacillus plantarum 16 (Lac16) and Paenibacillus polymyxa 10 (BSC10) against Clostridium perfringens (Cp) infection in broilers. A total of 720 one-day-old chicks were randomly divided into four groups. The control and Cp group were only fed a basal diet, while the two treatment groups received basal diets supplemented with Lac16 (1 × 108 cfu·kg-1) and BSC10 (1 × 108 cfu·kg-1) for 21 days, respectively. On day 1 and days 14 to 20, birds except those in the control group were challenged with 1 × 108 cfu C. perfringens type A strain once a day. The results showed that both Lac16 and BSC10 could ameliorate intestinal structure damage caused by C. perfringens infection. C. perfringens infection induced apoptosis by increasing the expression of Bax and p53 and decreasing Bcl-2 expression and inflammation evidence by higher levels of IFN-γ, IL-6, IL-1ß, iNOS, and IL-10 in the ileum mucosa, and NO production in jejunal mucosa, which was reversed by Lac16 and BSC10 treatment except for IL-1ß (P < 0.05). Besides, the two probiotics restored the intestinal microbiota imbalance induced by C. perfringens infection, characterized by the reduced Firmicutes and Proteobacteria and the increased Bacteroidetes at the phyla level and decreased Bacteroides fragilis and Gallibacterium anatis at the genus level. The two probiotics also reversed metabolic pathways of the microbiota in C. perfringens-infected broilers, including B-vitamin biosynthesis, peptidoglycan biosynthesis, and pyruvate fermentation to acetate and lactate II pathway. In conclusion, Lac16 and BSC10 can effectively protect broilers against C. perfringens infection through improved composition and metabolic pathways of the intestinal microbiota, intestinal structure, inflammation, and anti-apoptosis.