Cu-doped Fe3O4 nanoparticles for efficient detoxification of epsilon toxin: Toward substituting magnetically recyclable detoxifying agent for formaldehyde.

This research presents the synthesis and characterization of Cu-doped Fe3O4 (Cu-Fe3O4) nanoparticles as a magnetically recoverable and reusable detoxifying agent for the efficient and long-lasting neutralization of bacterial toxins. The nanoparticles were synthesized using the combustion synthesis method and characterized through SEM, XRD, BET, TGA, and VSM techniques. The detoxification potential of Cu-Fe3O4 was compared with traditional formaldehyde (FA) in detoxifying epsilon toxin (ETx) from Clostridium perfringens Type D, the causative agent of enterotoxemia in ruminants. In vivo residual toxicity tests revealed that Cu-Fe3O4 could detoxify ETx at a concentration of 2.0 mg mL-1 within 4 days at room temperature (RT) and 2 days at 37 °C, outperforming FA (12 and 6 days at RT and 37 °C, respectively). Characterization studies using dynamic light scattering (DLS) and circular dichroism (CD) highlighted lower conformational changes in Cu-Fe3O4-detoxified ETx compared to FA-detoxified ETx. Moreover, Cu-Fe3O4-detoxified ETx exhibited exceptional storage stability at 4 °C and RT for 6 months, maintaining an irreversible structure with no residual toxicity. The particles demonstrated remarkable reusability, with the ability to undergo five continuous detoxification batches. This study provides valuable insights into the development of an efficient and safe detoxifying agent, enabling the production of toxoids with a native-like structure. The magnetically recoverable and reusable nature of Cu-Fe3O4 nanoparticles offers practical advantages for easy recovery and reuse in detoxification reactions.

In silico mutation of aromatic with aliphatic amino acid residues in Clostridium perfringens epsilon toxin (ETX) reduces its binding efficiency to Caprine Myelin and lymphocyte (MAL) protein receptors.

Enterotoxaemia (ET) is a severe disease that affects domestic ruminants, including sheep and goats, and is caused by Clostridium perfringens type B and D strains. The disease is characterized by the production of Epsilon toxin (ETX), which has a significant impact on the farming industry due to its high lethality. The binding of ETX to the host cell receptor is crucial, but still poorly understood. Therefore, the structural features of goat Myelin and lymphocytic (MAL) protein were investigated and defined in this study. We induced the mutations in aromatic amino acid residues of ETX and substituted them with aliphatic residues at domains I and II. Subsequently, protein-protein interactions (PPI) were performed between ETX (wild)-MAL and ETX (mutated)-MAL protein predicting the domain sites of ETX structure. Further, molecular dynamics (MD) simulation studies were performed for both complexes to investigate the dynamic behavior of the proteins. The binding efficiency between 'ETX (wild)-MAL protein' and 'ETX (mutated)-MAL protein complex' interactions were compared and showed that the former had stronger interactions and binding efficiency due to the higher stability of the complex. The MD analysis showed destabilization and higher fluctuations in the PPI of the mutated heterodimeric ETX-MAL complex which is otherwise essential for its functional conformation. Such kind of interactions with mutated functional domains of ligands provided much-needed clarity in understanding the pre-pore complex formation of epsilon toxin with the MAL protein receptor of goats. The findings from this study would provide an impetus for designing a novel vaccine for Enterotoxaemia in goats.Communicated by Ramaswamy H. Sarma.

Use of Esienia fetida Worms to Produce Peptone for Clostridium perfringens Vaccine Production.

Concurrent with an increase in the human population on the earth, more than ever, the creation of energy and maintenance of health is necessary, and nowadays, various sources of energy supply are being developed. The general global view in this regard is to provide protein and energy from available and cheap sources. Iran is no exception to this general rule, only in the field of ensuring the health of livestock resources every year, about 10 tons of peptone is needed for producing clostridial vaccines. Vermicomposting worms (Esienia fetida) with high protein percentages and rapid reproductions are a suitable source for peptone production. Based on this, the vaccine strain of Clostridium perfringens type D cultivated in two different media contain peptone produced from worms and meat peptone. The growth rate, epsilon toxin (ETX), and alpha toxin (CPA) of Cl. perfringens have been compared in two media. The results showed that the growth rate of bacteria in the worm peptone medium in 48 h was 22% higher than that of the meat peptone. Additionally, the activity of alpha toxin (phospholipase C) was in worm peptone 15% higher than meat peptone during 80 min of measurement. Regarding epsilon toxin lethality, all three mice of the N-worm peptone group died, while all three mice of the meat peptone group survived even 72 h after injection. The average survival time of mice in the N-worm peptone group was 1700 min. Therefore, we suggest the worms' protein is more suitable than industrial meat in peptone production for vicinal propose. To eliminate the need for hydrolyzed protein in the production of vaccines in the future, we suggest an increase in the fields of employment and the development of fertilizer and worm farms in Iran.

Development and evaluation of cell membrane-based biomimetic nanoparticles loaded by Clostridium perfringens epsilon toxin: a novel vaccine delivery platform for Clostridial-associated diseases.

As Clostridium perfringens (C. perfringens) epsilon toxin (ETX) ranks as the third most potent clostridial toxin after botulinum and tetanus toxins, vaccination is necessary for creatures that can be affected by it to be safe from the effects of this toxin. Nowadays, nanostructures are good choices for carriers for biological environments. We aimed to synthesize biomimetic biodegradable nanodevices to enhance the efficiency of the ETX vaccine. For this purpose, poly(lactic-co-glycolic acid) (PLGA) copolymer loaded with purified epsilon protoxin (proETX) to create nanoparticles called nanotoxins (NTs) and then coated by RBC membrane-derived vesicles (RVs) to form epsilon nanotoxoids (RV-NTs). The resulting RV-NTs shaped smooth spherical surfaces with double-layer core/shell structure with an average particle size of 105.9 ± 35.1 nm and encapsulation efficiency of 97.5% ± 0.13%. Compared with NTs, the RV-NTs were more stable for 15 consecutive days. In addition, although both structures showed a long-term cumulative release, the release rates from RV-NTs were slower than NTs during 144 hours. According to the results of cell viability, ETX loading in PLGA and entrapment in the RBC membrane decreased the toxicity of the toxin. The presence of PLGA enhances the uptake of proETX, and the synthesized structures showed no significant lesion after injection. These results demonstrate that NTs and RV-NTs could serve as an effective vaccine platform to deliver ETX for future in vivo assays.

Optimization and scale-up of Clostridium perfringens type D culture and epsilon-toxin production: Effects of stirring, glucose and pH adjustment.

The effects of some main bacteria culture parameters including mixing rate, glucose (GC) concentration, steps of GC addition, and steps of pH adjustment on both C. perfringens bacteria growth and its epsilon toxin production in a bench-scale 20-L glass carboy were investigated. The optimized mixing rate of 300 rpm, GC concentration of 4 g L-1, and 3-step addition of GC resulted in the bacteria and toxin concentrations of 0.16 g L-1 and 330 ng mL-1, respectively. Also, the induction of a pH shock at the reaction time of 180 min led to the remarkable enhancement of toxin production (367 ng mL-1). Upon applying both optimized conditions for GC addition and pH adjustment, the high toxin concentration of 433 ng mL-1 was obtained. Using the constant mixing rate technique, the process was scaled up to a 1500-L industrial bioreactor, where its performance was close to the bench-scale bioreactor (i.e., toxin concentration of 419 ng mL-1). The results revealed the reliability of this method to economically improve and scale up the bacteria culture process, which can be further used for other microbial fermentations.

Is Clostridium perfringens epsilon toxin associated with multiple sclerosis?

Clostridium perfringens epsilon toxin is associated with enterotoxaemia in livestock. More recently, it is proposed to play a role in multiple sclerosis (MS) in humans. Compared to matched controls, strains of C. perfringens which produce epsilon toxin are significantly more likely to be isolated from the gut of MS patients and at significantly higher levels; similarly, sera from MS patients are significantly more likely to contain antibodies to epsilon toxin. Epsilon toxin recognises the myelin and lymphocyte (MAL) protein receptor, damaging the blood-brain barrier and brain cells expressing MAL. In the experimental autoimmune encephalomyelitis model of MS, the toxin enables infiltration of immune cells into the central nervous system, inducing an MS-like disease. These studies provide evidence that epsilon toxin plays a role in MS, but do not yet fulfil Koch's postulates in proving a causal role.

Clostridium perfringens Epsilon Toxin Binds to and Kills Primary Human Lymphocytes.

Clostridium perfringens epsilon toxin (ETX) is the third most lethal bacterial toxin and has been suggested to be an environmental trigger of multiple sclerosis, an immune-mediated disease of the human central nervous system. However, ETX cytotoxicity on primary human cells has not been investigated. In this article, we demonstrate that ETX preferentially binds to and kills human lymphocytes expressing increased levels of the myelin and lymphocyte protein MAL. Using flow cytometry, ETX binding was determined to be time and dose dependent and was highest for CD4+ cells, followed by CD8+ and then CD19+ cells. Similar results were seen with ETX-induced cytotoxicity. To determine if ETX preference for CD4+ cells was related to MAL expression, MAL gene expression was determined by RT-qPCR. CD4+ cells had the highest amount of Mal gene expression followed by CD8+ and CD19+ cells. These data indicate that primary human cells are susceptible to ETX and support the hypothesis that MAL is a main receptor for ETX. Interestingly, ETX bindings to human lymphocytes suggest that ETX may influence immune response in multiple sclerosis.

PLGA Nanoparticle-Based Dissolving Microneedle Vaccine of Clostridium perfringens ε Toxin.

Epsilon toxin (ETX) is an exotoxin produced by type B and D Clostridium perfringens that causes enterotoxemia or necrotic enteritis in animals such as goats, sheep, and cattle. Vaccination is a key method in preventing such diseases. In this study, we developed a new type of dissolving microneedle patch (dMN) with a nanoparticle adjuvant for enhanced immune response to deliver the rETXY196E-C protein vaccine. We chose FDA-approved poly(lactic-co-glycolic acid) (PLGA) to prepare nanospheres as the vaccine adjuvant and introduced dimethyldioctadecylammonium bromide (DDAB) to make the surface of PLGA nanoparticles (PLGA NPs) positively charged for antigen adsorption. PLGA NPs with a diameter of 100~200 nm, a surface ZETA potential of approximately +40 mV, and good safety were successfully prepared and could effectively adsorb rETXY196E-C protein. Using non-toxic and antibacterial fish gelatin as the microneedle (MN) matrix, we prepared a PLGA-DDAB dMN vaccine with good mechanical properties that successfully penetrated the skin. After immunization of subcutaneous (SC) and dMN, antibody titers of the PLGA and Al adjuvant groups were similar in both two immune ways. However, in vivo neutralization experiments showed that the dMN vaccines had a better protective effect. When challenged with 100 × LD50 GST-ETX, the survival rate of the MN group was 100%, while that of the SC Al group was 80%. However, a 100% protective effect was achieved in both immunization methods using PLGA NPs. In vitro neutralization experiments showed that the serum antibodies from the dMN and SC PLGA NPs groups both protect naive mice from 10 × LD50 GST-ETX attack after being diluted 20 times and could also protect MDCK cells from 20 × CT50 GST-ETX attack. In conclusion, the PLGA-DDAB dMN vaccine we prepared has good mechanical properties, immunogenicity, and protection, and can effectively prevent ETX poisoning. This provides a better way of delivering protein vaccines.

Clostridium perfringens epsilon-toxin requires acid sphingomyelinase for cellular entry.

OBJECTIVES: Clostridium perfringens epsilon-toxin is considered to be a crucial agent in enterotoxemia in domestic animals. Epsilon-toxin enters host cells via endocytosis and results in the formation of late endosome/lysosome-derived vacuoles. In the present study, we found that acid sphingomyelinase promotes the internalization of epsilon-toxin in MDCK cells. METHODS: We measured the extracellular release of acid sphingomyelinase (ASMase) by epsilon-toxin. We examined the role of ASMase in epsilon-toxin-induced cytotoxicity using selective inhibitors and knockdown of ASMase. Production of ceramide after toxin treatment was determined by immunofluorescence technique. RESULTS: Blocking agents of ASMase and exocytosis of lysosomes inhibited this epsilon-toxin-induced vacuole formation. Lysosomal ASMase was liberated to extracellular space during treatment of the cells with epsilon-toxin in the presence of Ca2+. RNAi-mediated attenuation of ASMase blocked epsilon-toxin-induced vacuolation. Moreover, incubation of MDCK cells with epsilon-toxin led to production of ceramide. The ceramide colocalized with lipid raft-binding cholera toxin subunit B (CTB) in the cell membrane, indicating that conversion of lipid raft associated sphingomyelin to ceramide by ASMase facilitates lesion of MDCK cells and internalization of epsilon-toxin. CONCLUSIONS: Based on the present results, ASMase is required for efficient internalization of epsilon-toxin.

Enterotoxemia produced by lambda toxin-positive Clostridium perfringens type D in 2 neonatal goat kids.

Enterotoxemia caused by Clostridium perfringens type D usually affects sheep and goats ≥ 2-wk-old. The main clinical signs and lesions of the disease are produced by the epsilon toxin (ETX) elaborated by this microorganism. However, ETX is produced in the form of a mostly inactive prototoxin that requires protease cleavage for activation. It has traditionally been believed that younger animals are not affected by type D enterotoxemia given the low trypsin activity in the intestinal content associated with the trypsin-inhibitory action of colostrum. Two Nigerian dwarf goat kids, 2- and 3-d-old, with a history of acute diarrhea followed by death, were submitted for postmortem examination and diagnostic workup. Autopsy and histopathology revealed mesocolonic edema, necrosuppurative colitis, and protein-rich pulmonary edema. Alpha toxin and ETX were detected in intestinal content, and C. perfringens type D was isolated from the colon of both animals. The isolates encoded the gene for lambda toxin, a protease that has been shown previously to activate ETX in vitro. Type D enterotoxemia has not been reported previously in neonatal kids, to our knowledge, and we suggest that lambda toxin activated the ETX.

Direct Visualization of the Dynamic Process of Epsilon Toxin on Hemolysis.

Hemolysis is the process of rupturing erythrocytes (red blood cells) by forming nanopores on their membranes using hemolysins, which then impede membrane permeability. However, the self-assembly process before the state of transmembrane pores and underlying mechanisms of conformational change are not fully understood. In this work, theoretical and experimental evidence of the pre-pore morphology of Clostridium perfringens epsilon toxin (ETX), a typical hemolysin, is provided using in situ atomic force microscopy (AFM) complemented by molecular dynamics (MD) simulations to detect the conformational distribution of different states in Mica. The AFM suggests that the ETX pore is formed in two stages: ETX monomers first attach to the membrane and form a pre-pore in no special conditions required, which then undergo a conformational change to form a transmembrane pore at temperatures above the critical point in the presence of receptors. The authors' MD simulations reveal that initial nucleation occurs when specific amino acids adsorb to negatively charged mica cavities. This work fills the knowledge gap in understanding the early stage of hemolysis and the oligomerization of hemolysins. Moreover, the newly identified pre-pore of ETX holds promise as a candidate for nanopore applications.

Clostridium perfringens Type D Epsilon Toxin Causes Blood-Retinal Barrier Microvascular Damage and Diffuse Retinal Vasogenic Oedema.

Clostridium perfringens type D epsilon toxin (ETX) causes severe retinal microvascular endothelial injury in the rat. The resulting blood-retinal barrier (BRB) breakdown leads to increased vascular permeability, which was detected immunohistochemically by the extravasation of plasma albumin as a vascular tracer, and ensuing severe, diffuse, vasogenic retinal oedema. This microvascular damage was also confirmed by a loss of endothelial barrier antigen, a marker of an intact BRB in rats. Since similar microvascular lesions are found in EXT-exposed laboratory rodent and sheep brains, and the BRB resembles the BBB, they are also likely to occur in the eyes of naturally epsilon-intoxicated sheep and goats, but this remains to be determined. Moreover, while retinal oedema is a common and important component of many human and veterinary ocular disorders, more effective treatments are required. Accordingly, the retinal vasogenic oedema reliably and reproducibly induced by ETX in rats provides a useful model in which to study the pathogenesis of retinal oedema development and evaluate its prevention or amelioration by putative pharmacological interventions.

Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for Detection of Epsilon Toxin.

Background: Epsilon toxin (ETX), produced by Clostridium perfringens, is one of the most potent toxins known, with a lethal potency approaching that of botulinum neurotoxins. Epsilon toxin is responsible for enteritis. Therefore, the development of rapid and simple methods to detect ETX is imperative. Aptamers are single-stranded oligonucleotides that can bind tightly to specific target molecules with an affinity comparable to that of monoclonal antibodies (mAbs). DNA aptamers can serve as tools for the molecular identification of organisms, such as pathogen subspecies. Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers against ETX. Methods: This study identified aptamers using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, enzyme-linked apta-sorbent assay (ELASA), and surface plasmon resonance (SPR) to determine the affinity and specificity of the newly obtained aptamers targeting ETX. Results: Several aptamers obtained through the SELEX process were studied. Among them, 2 aptamers, ETX clone 3 (ETX3; dissociation constant (Kd = 8.4 ± 2.4E-9M) and ETX11 (Kd = 6.3 ± 1.3E-9M) had favorable specificity for ETX. The limits of detection were 0.21 and 0.08 µg/mL for ETX3 and ETX11, respectively. . Conclusions: The discovered aptamers can be used in various aptamer-based rapid diagnostic tests for the detection of ETX.

Interaction of Clostridium perfringens Epsilon Toxin with the Plasma Membrane: The Role of Amino Acids Y42, Y43 and H162.

Clostridium perfringens epsilon toxin (Etx) is a pore forming toxin that causes enterotoxaemia in ruminants and may be a cause of multiple sclerosis in humans. To date, most in vitro studies of Etx have used the Madin-Darby canine kidney (MDCK) cell line. However, studies using Chinese hamster ovary (CHO) cells engineered to express the putative Etx receptor, myelin and lymphocyte protein (MAL), suggest that amino acids important for Etx activity differ between species. In this study, we investigated the role of amino acids Y42, Y43 and H162, previously identified as important in Etx activity towards MDCK cells, in Etx activity towards CHO-human MAL (CHO-hMAL) cells, human red blood cells (hRBCs) and synthetic bilayers using site-directed mutants of Etx. We show that in CHO-hMAL cells Y42 is critical for Etx binding and not Y43 as in MDCK cells, indicating that surface exposed tyrosine residues in the receptor binding domain of Etx impact efficiency of cell binding to MAL-expressing cells in a species-specific manner. We also show that Etx mutant H162A was unable to lyse CHO-hMAL cells, lysed hRBCs, whilst it was able to form pores in synthetic bilayers, providing evidence of the complexity of Etx pore formation in different lipid environments.

Pathology and Pathogenesis of Brain Lesions Produced by Clostridium perfringens Type D Epsilon Toxin.

Clostridium perfringens type D epsilon toxin (ETX) produces severe, and frequently fatal, neurologic disease in ruminant livestock. The disorder is of worldwide distribution and, although vaccination has reduced its prevalence, ETX still causes substantial economic loss in livestock enterprises. The toxin is produced in the intestine as a relatively inactive prototoxin, which is subsequently fully enzymatically activated to ETX. When changed conditions in the intestinal milieu, particularly starch overload, favor rapid proliferation of this clostridial bacterium, large amounts of ETX can be elaborated. When sufficient toxin is absorbed from the intestine into the systemic circulation and reaches the brain, two neurologic syndromes can develop from this enterotoxemia. If the brain is exposed to large amounts of ETX, the lesions are fundamentally vasculocentric. The neurotoxin binds to microvascular endothelial receptors and other brain cells, the resulting damage causing increased vascular permeability and extravasation of plasma protein and abundant fluid into the brain parenchyma. While plasma protein, particularly albumin, pools largely perivascularly, the vasogenic edema becomes widely distributed in the brain, leading to a marked rise in intracranial pressure, coma, sometimes cerebellar herniation, and, eventually, often death. When smaller quantities of ETX are absorbed into the bloodstream, or livestock are partially immune, a more protracted clinical course ensues. The resulting brain injury is characterized by bilaterally symmetrical necrotic foci in certain selectively vulnerable neuroanatomic sites, termed focal symmetrical encephalomalacia. ETX has also been internationally listed as a potential bioterrorism agent. Although there are no confirmed human cases of ETX intoxication, the relatively wide species susceptibility to this toxin and its high toxicity mean it is likely that human populations would also be vulnerable to its neurotoxic actions. While the pathogenesis of ETX toxicity in the brain is incompletely understood, the putative mechanisms involved in neural lesion development are discussed.

Cellular effects of epsilon toxin on the cell viability and oxidative stress of normal and lung cancer cells.

INTRODUCTION: Clostridium perfringens is a type of gram-positive anaerobic bacilli. C.perfringens produces many toxins, of which epsilon (ε) is one of the major ones. The mechanism of epsilon's toxicity is located in the lipid of cell membrane tissues. Epsilon toxin is known as a bioterrorism agent. Inhalation of these aerosols can destroy pulmonary vascular endothelial cells and cause lung injury, which increases vascular permeability and pulmonary edema. METHODS: In this study, we investigated the toxicity of epsilon toxin by using the MTT assay, evaluated oxidative stress effects such as ROS and LPO using the DCFH and TBA reagents, and measured the GSH of the normal and lung cancer cells by using the DTNB reagent. RESULTS: The result showed that 1 µg/ml of epsilon toxin caused mitochondrial disorder and reduced the growth of the normal cell line. This toxin also induced ROS and damage to lipid membranes. Furthermore, the same effect occurred in the lung cancer cell, and the epsilon toxin inhibited cancer cell proliferation. CONCLUSION: This toxin causes toxicity by binding to lipid membranes. As the present study results have confirmed, epsilon toxin inhibits mitochondrial function and induces ROS and lipid membrane damage.

Enterocolitis in goats associated with enterotoxaemia in the perspective of two toxins: Epsilon toxin and beta-2 toxin - An immunohistochemical and molecular study.

Caprine intestinal diseases associated with clostridia are generally caused by Cpa and Etx encoded alpha (α) and epsilon (ε) toxinotypes of Clostridium perfringens type D respectively. A recent study on goat enterocolitis, demonstrated that the incidence of Clostridium perfringens type-D toxinotype and beta 2 toxins is high, suggesting its role in enterocolitis and many other diseases of goats affecting intestinal tract. Considering this scenario, the present prevalence study was planned to screen the goat intestinal tissues for the presence of the epsilon toxin and beta 2 toxin. Tissue sections from enterotoxaemia suspected cases in 189 goats were collected and epsilon-toxin was demonstrated by immuno-histochemically and toxinotyping multiplex polymerase reaction in 19 animals and beta 2 toxin in 19 animals by multiplex polymerase reaction. Immuno-reactivity to epsilon toxin was detected maximum in distal ileum of goat intestine and this toxin was produced by Clostridium perfringens type D. It suggests that immunohistochemistry is a confirmatory tool for detection of bacterial toxin especially epsilon toxin where isolation and characterisation of bacteria is not possible. Here, we have reported a strong association between ε-toxin (epsilon) and beta-2 toxin in causing disorders of intestine in goats. In addition, we have explored the possible role of cpb2 positive isolates of C. perfringens and their pathogenic effects in causing enterotoxaemia. These determinants help in the understanding of the pathogenesis of enterotoxaemia in goats which needs to be further investigated.

New Mutants of Epsilon Toxin from Clostridium perfringens with an Altered Receptor-Binding Site and Cell-Type Specificity.

Epsilon toxin (Etx) from Clostridium perfringens is the third most potent toxin after the botulinum and tetanus toxins. Etx is the main agent of enterotoxemia in ruminants and is produced by Clostridium perfringens toxinotypes B and D, causing great economic losses. Etx selectively binds to target cells, oligomerizes and inserts into the plasma membrane, and forms pores. A series of mutants have been previously generated to understand the cellular and molecular mechanisms of the toxin and to obtain valid molecular tools for effective vaccination protocols. Here, two new non-toxic Etx mutants were generated by selective deletions in the binding (Etx-ΔS188-F196) or insertion (Etx-ΔV108-F135) domains of the toxin. As expected, our results showed that Etx-ΔS188-F196 did not exhibit the usual Etx binding pattern but surprisingly recognized specifically an O-glycoprotein present in the proximal tubules of the kidneys in a wide range of animals, including ruminants. Although diminished, Etx-ΔV108-F135 maintained the capacity for binding and even oligomerization, indicating that the mutation particularly affected the pore-forming ability of the toxin.

Heterogeneous immunoreactivity of axonal spheroids in focal symmetrical encephalomalacia produced by Clostridium perfringens type D epsilon toxin in sheep.

Since axonal injury (AI) is an important component of many veterinary neurologic disorders, we assessed the relative ability of a panel of antibodies (amyloid precursor protein, 3 subunits of neurofilament protein, protein gene product 9.5, ubiquitin, and synaptophysin) to detect axonal swellings or spheroids. Abundant axonal spheroids found in necrotic internal capsule foci produced in 4 sheep by chronic Clostridium perfringens type D epsilon neurotoxicity provided a model system in which to evaluate this important diagnostic tool. There was heterogeneous labeling of subsets of spheroids by the respective antibodies, suggesting that, in order to detect the complete spectrum of AI in diagnostic cases, a range of antibodies should be used, not only when spheroids are plentiful but also when they are few in number or incompletely developed. The application of insufficient markers in the latter cases can potentially lead to the contribution of AI to lesion pathogenesis being underappreciated.

A simple method for purification of epsilon toxin of Clostridium perfringens type D for serum neutralization assay.

Enterotoxaemia, a disease that affects domestic ruminants, is caused by the epsilon toxin of Clostridium perfringens type D and B. Control and prophylaxis are based on systemic vaccination of small ruminant herds with epsilon toxoid. Purified epsilon toxin is an essential material for vaccine evaluation. It is also necessary for diagnosis of enterotoxaemia disease in the field by in vitro tests including ELISA. The aim of this study was to set up a method for preparation of functional purified epsilon toxin of C. perfringens type D to be used in serum neutralization test. In this study, epsilon toxin was prepared from C. perfringens type D culture precipitated with ammonium sulfate, dialyzed against phosphate buffered saline (PBS) buffer and then, purified using chromatography system. Then, the purified epsilon toxin was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Toxin function was confirmed by cell culture and minimum lethal dose (MLD) assays. Also rabbits were immunized by vaccine in two turns with a 28-day interval. Then, blood samples were collected, and serum neutralization (SN) test was carried out. Results showed that the purified toxin was suitable for SN assay. Our purification method was simple, fast and cost-effective for preparation of epsilon toxin.