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 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.

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.

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.

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.

Investigation of genetic toxicity and oxidative stress of Clostridium perfringens epsilon toxin type D on human peripheral blood lymphocytes.

Epsilon toxin (Etx) is an enormously potent pore-forming toxin and a category B biological agent. Etx is the main virulence determinant of Clostridiumperfringens types B and D toxin. It has a cytotoxic effect on distal and collecting kidney tubules. Also, Etx crosses the blood-brain barrier, binds to myelin structures, and destroys oligodendrocytes. The main purpose of this study was to investigate the toxic effects of Etx on human blood lymphocytes, which we examined for the first time for the genetic toxicity of this bacterial toxin. In this study, after taking blood and dividing into nine groups and putting in contact with different dilutions of Etx (1,5,10,25,50,100 and 200 µM), methotrexate (750 µM), and normal saline by Cytokinesis blocked micronucleus (CBMN) assay, we looked at genetic toxicity and the level of oxidative stress created in the under study lymphocytes. The results of this study showed that Etx has significant oxidative stress effects on human lymphocytes at doses above 25 µM, and also this bacterial toxin significantly increases the number of micronuclei formed in lymphocytes. The results of this study indicate that Etx has toxic effects it is genetic and interferes with cell division processes. Thus, human lymphocytes can be used extensively in future studies on Etx.

Cardiopulmonary Lesions in Sheep Produced by Experimental Acute Clostridium Perfringens Type D Enterotoxemia.

Enterotoxemia caused by Clostridium perfringens type D is one of the most prevalent clostridial diseases of sheep. The lesions of the acute form of this disease, particularly the cerebral lesions, are well characterized; however, detailed descriptions of the cardiac and pulmonary lesions are lacking. Here we describe cardiopulmonary lesions in experimental acute type D enterotoxemia in sheep and determine the role of epsilon toxin (ETX) in the development of these lesions. Four groups of 6 sheep were intraduodenally inoculated with either a wild-type C. perfringens type D strain; its etx knockout mutant, which is unable to produce ETX; the etx mutant complemented with the wild-type etx gene, which regains the ETX toxigenic ability; or sterile culture medium as a control. All sheep were subjected to postmortem examination within 24 hours of inoculation. Lesion scores were compared between groups for pulmonary edema; hydrothorax; ascites; hydropericardium; endocardial, myocardial and epicardial hemorrhages; microscopic lesions of acute myocardial degeneration and necrosis; and myocardial, endocardial, and epicardial edema, hemorrhage, and inflammation. Only sheep inoculated with the wild-type and complemented ETX-toxigenic bacterial strains developed cardiopulmonary lesions, which were present in varying degrees of severity and proportions. These lesions were not present in sheep inoculated with the etx mutant or in the negative control. We conclude that severe acute cardiopulmonary lesions in sheep with experimental enterotoxemia are associated with the capacity of the strains to produce ETX. These changes are likely contributors to the clinical signs and even death of affected animals.

Clostridium perfringens epsilon toxin binds to erythrocyte MAL receptors and triggers phosphatidylserine exposure.

Epsilon toxin (ETX) is a 33-kDa pore-forming toxin produced by type B and D strains of Clostridium perfringens. We previously found that ETX caused haemolysis of human red blood cells, but not of erythrocytes from other species. The cellular and molecular mechanisms of ETX-mediated haemolysis are not well understood. Here, we investigated the effects of ETX on erythrocyte volume and the role of the putative myelin and lymphocyte (MAL) receptors in ETX-mediated haemolysis. We observed that ETX initially decreased erythrocyte size, followed by a gradual increase in volume until lysis. Moreover, ETX triggered phosphatidylserine (PS) exposure and enhanced ceramide abundance in erythrocytes. Cell shrinkage, PS exposure and enhanced ceramide abundance were preceded by increases in intracellular Ca2+ concentration. Interestingly, lentivirus-mediated RNA interference studies in the human erythroleukaemia cell line (HEL) cells confirmed that MAL contributes to ETX-induced cytotoxicity. Additionally, ETX was shown to bind to MAL in vitro. The results of this study recommend that ETX-mediated haemolysis is associated with MAL receptor activation in human erythrocytes. These data imply that interventions affecting local MAL-mediated autocrine and paracrine signalling may prevent ETX-mediated erythrocyte damage.

Development of a double-recombinant antibody sandwich ELISA for quantitative detection of epsilon toxoid concentration in inactivated Clostridium perfringens vaccines.

BACKGROUND: Epsilon toxin (ETX) causes a commonly fatal enterotoxemia in domestic animals. Also, ETX causes serious economic losses to animal husbandry. In this study, we selected several clones against ETX using repertoires displayed on filamentous phage. Anti-ETX specific clones were enriched by binding to immobilized antigen, followed by elution and re-propagation of phage. After multiple rounds of binding selection, ELISA analysis showed that most isolated clones had high affinity and specificity for ETX. RESULTS: Two recombinant monoclonal antibodies against ETX were isolated by phage display technology. B1 phage VH antibody isolated from DAb library and G2 soluble scFv antibody isolated from Tomlinson I + J libraries have been applied as the capture and detection antibodies for developing an ETX sandwich ELISA test, respectively. CONCLUSIONS: Designed ETX sandwich ELISA could be a valuable tool for quantitative detection of ETX in inactivated commercial vaccines against enterotoxemia.

Putative serum protein biomarkers for epsilon toxin exposure in mouse model using LC-MS/MS analysis.

Epsilon toxin (ETX), produced by Clostridium perfringens Type B or type D strains, is a potential biological and toxin warfare (BTW) agent, largely for its very high toxicity. The toxin is implicated in several animal diseases. Using LC-MS/MS analysis, we report here elucidation of putative serum maker proteins for ETX exposure with an objective of the early diagnosis of intoxication. Of 166 consensus proteins (488 peptides), showing ETX-induced alterations, 119 proteins exhibited increase and 47 proteins showed decreased abundance in serum, as revealed by SWATH (DIA) acquisition on LC-MS/MS and label free quantitative analysis of control and test samples. Complement and coagulation cascade, nitrogen metabolism, negative regulation of peptidase activity, and response to ROS were among the biological processes and pathways perturbed by the ETX exposure. Interaction network indicated enzyme inhibitor activity, detoxification of ROS, and steroid binding functions were the major interaction networks for the proteins with increased abundance, while, hemostasis and structural molecule activity were the prominent networks for the down-regulated proteins. Validation studies were carried out by immunoprecipitation, ELISA, and Western blot analysis of selected proteins to demonstrate diagnostic potential of the putative marker proteins of ETX exposure.

Death of a neonatal lamb due to Clostridium perfringens type B in New Zealand.

Case history and clinical findings: A flock of 20 sheep was kept within three paddocks on a single property. None of the animals in the flock had been vaccinated against any disease for at least three years. Abdominal bloating and haemorrhagic diarrhoea were observed in Lamb 1 at 24 hours-of-age. The lamb subsequently died within an hour of the onset of clinical signs. Lamb 2 was 3-days-old when observed to be recumbent with opisthotonus. The lamb was treated with dextrose, vitamins B1 and B12, and penicillin G, but died 4 hours later.Pathological findings: Examination of Lamb 1 revealed markedly increased gas within the peritoneum and within dilated loops of intestine. The intestines were dark red and contained large quantities of haemorrhagic fluid. Histology of the intestines revealed peracute mucosal necrosis with minimal accompanying inflammation. The intestinal lumen contained cell debris, haemorrhage, and myriad large Gram-positive bacilli. The intestines of Lamb 2 did not appear bloated or reddened. However, multiple fibrin clots were visible within the pericardial sac. Histopathological examination revealed small foci of necrosis within the mucosa of the distal intestine. The necrotic foci were often associated with large numbers of large Gram-positive bacilli.Immunohistochemsitry and molecular biology: Intestinal samples from Lamb 1 were processed for Clostridium perfringens immunohistochemistry, which revealed large numbers of intralesional, positively immunostained rods. Fragments corresponding to the expected sizes for genes encoding alpha, beta, and epsilon C. perfringens typing toxins were amplified by PCR from DNA extracted from formalin-fixed sections of intestine.Diagnosis: Lamb dysentery due to C. perfringens type B.Clinical relevance: C. perfringens bacteria have a worldwide distribution, but disease due to C. perfringens type B has only been diagnosed in a small number of countries and has never been reported in New Zealand or Australia. C. perfringens type B produce both beta toxin and epsilon toxins, therefore both haemorrhagic enteritis and systemic vascular damage can develop. As many animals are exposed to C. perfringens without developing disease, there must be additional unknown factors that resulted in disease in these particular sheep. Vaccines that specifically protect against C. perfringens type B are available and may be recommended for use in smaller non-commercial flocks, as in the present case.

Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis.

BACKGROUND: It was recently reported that, using Western blotting, some multiple sclerosis (MS) patients in the United States had antibodies against epsilon toxin (Etx) from Clostridium perfringens, suggesting that the toxin may play a role in the disease. OBJECTIVE: We investigated for serum antibodies against Etx in UK patients with clinically definite multiple sclerosis (CDMS) or presenting with clinically isolated syndrome (CIS) or optic neuritis (ON) and in age- and gender-matched controls. METHODS: We tested sera from CDMS, CIS or ON patients or controls by Western blotting. We also tested CDMS sera for reactivity with linear overlapping peptides spanning the amino acid sequence (Pepscan) of Etx. RESULTS: Using Western blotting, 24% of sera in the combined CDMS, CIS and ON groups ( n = 125) reacted with Etx. In the control group ( n = 125), 10% of the samples reacted. Using Pepscan, 33% of sera tested reacted with at least one peptide, whereas in the control group only 16% of sera reacted. Out of 61 samples, 21 (43%) were positive to one or other testing methodology. Three samples were positive by Western blotting and Pepscan. CONCLUSION: Our results broadly support the previous findings and the role of Etx in the aetiology of MS warrants further investigation.

Elucidation of protein biomarkers in plasma and urine for epsilon toxin exposure in mouse model.

Epsilon toxin (ETX) is the major virulence determinant of C. perfringens type B or type D strains, causing diseases in animals, besides being a listed biological and toxin warfare (BTW) agent. Keeping in mind the high lethality and the rapid onset of clinical manifestations, early diagnosis of epsilon toxin exposure is of paramount importance for implementation of appropriate medical countermeasures. Using a 2DE-MS approach, the present study is the first comprehensive proteomic elucidation of ETX-induced protein markers in the mouse model, providing putative targets for early diagnosis of ETX exposure. A total of 52 unique proteins showing ETX-induced modulations were identified in plasma and urine samples. Fibrinogen, apolipoprotein, serum amyloid protein, plasminogen, serum albumin, glutathione peroxidase, transferrin, major urinary protein 2, haptoglobin, transthyretin, and vitamin D-binding protein were among the proteins observed in more than one dataset with altered abundance after the ETX-intoxication. The predicted localization, function, and interaction of the ETX-modulated proteins in the plasma and urine indicated involvement of multiple pathways; extracellular proteins, followed by macromolecular complexes associated with blood coagulation and plasminogen activating cascade, being the most prominent among others. The putative markers elucidated here warrants further validation and can be of immense value for the early diagnosis of ETX exposure.

Synthesis of peptide based epsilon toxin vaccine by covalent anchoring to tetanus toxoid.

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.

Epsilon toxin from Clostridium perfringens induces cytotoxicity in FRT thyroid epithelial cells.

Epsilon toxin (Etx) is produced by Clostridium perfringens and induces enterotoxemia in ruminants. Etx crosses the blood-brain barrier, binds to myelin structures, and kills oligodendrocytes, inducing central nervous system demyelination. In addition, Etx has a cytotoxic effect on distal and collecting kidney tubules. There are few cell lines sensitive to Etx. At present, the most sensitive in vitro model for Etx is the Madin-Darby canine kidney (MDCK) cell line, where Etx oligomerizes and forms a pore with consequent ion efflux and cell death. Although the Etx receptor has not yet been fully clarified, it is known that caveolin 1 and 2 potentiate Etx cytotoxicity and oligomerization, and more recently, the myelin and lymphocyte (MAL) protein has been implicated in Etx binding and activity. Here, we studied the effect of Etx on Fischer rat thyroid cells (FRT) and observed similar effects as those seen in MDCK cells. Etx incubated with FRT cells showed binding to the plasma membrane, and western blotting assays revealed oligomeric complex formation. Moreover, cytotoxic assays on FRT cells after Etx incubation indicated cell death at a similar level as in MDCK cells. In addition, a luminescent ATP detection assay revealed ATP depletion in FRT cells after Etx exposure. Previous studies have reported that FRT cells do not express caveolins and do not form caveolae but express MAL protein in glycolipid-enriched membrane microdomains. Our results indicate that caveolins are not directly implicated in Etx cytotoxicity, supporting the notion that the MAL protein is involved in Etx action. In addition, a cell line of thyroid origin is described for the first time as a good model to study Etx action.

Identification of epsilon toxin-producing Clostridium perfringens strains in American retail food.

Food samples (n = 216) from New York city were tested for the presence of C. perfringens via PCR for specific toxin genes. Thirty-four (16%) samples were positive for C. perfringens. Of these 34, 31 (91.2%) were type A or E, one (2.9%) was type B, and two (5.9%) were type D.

Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral sphingomyelinase.

BACKGROUND: Clostridium perfringens epsilon-toxin is responsible for fatal enterotoxemia in ungulates. The toxin forms a heptamer in the lipid rafts of Madin-Darby Canine Kidney (MDCK) cells, leading to cell death. Here, we showed that epsilon-toxin requires neutral sphingomyelinase (nSMase) activity during oligomerization. METHODS: We tested the role of nSMase in the oligomerization of epsilon-toxin using specific inhibitors, knockdown of nSMase, formation of ceramide, and localization of epsilon-toxin and ceramide by immunofluorescence staining. RESULTS: Epsilon-toxin induced the production of ceramide is a dose- and time-dependent manner in ACHN cells. GW4869, an inhibitor of nSMase, inhibited ceramide production induced by the toxin. GW4869 and knockdown of nSMase blocked toxin-induced cell death and oligomer formation of epsilon-toxin. Confocal microscopy images showed that the toxin induced ceramide clustering and colocalized with ceramide. CONCLUSIONS: These results demonstrated that oligomer formation of epsilon-toxin is facilitated by the production of ceramide through activation of nSMase caused by the toxin. GENERAL SIGNIFICANCE: Inhibitors of nSMase may confer protection against infection.

Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells.

Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a ß-barrel of 14 amphipathic ß-strands. ETX shows a high specificity for certain cell lines, of which Madin-Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes.

New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia.

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is responsible for diseases that occur mostly in ruminants. ETX is produced in the form of an inactive prototoxin that becomes proteolytically-activated by several proteases. A recent ex vivo study using caprine intestinal contents demonstrated that ETX prototoxin is processed in a step-wise fashion into a stable, active ∼27 kDa band on SDS-PAGE. When characterized further by mass spectrometry, the stable ∼27 kDa band was shown to contain three ETX species with varying C-terminal residues; each of these ETX species is cytotoxic. This study also demonstrated that, in addition to trypsin and chymotrypsin, proteases such as carboxypeptidases are involved in processing ETX prototoxin. Once absorbed, activated ETX species travel to several internal organs, including the brain, where this toxin acts on the vasculature to cross the blood-brain barrier, produces perivascular edema and affects several types of brain cells including neurons, astrocytes, and oligodendrocytes. In addition to perivascular edema, affected animals show edema within the vascular walls. This edema separates the astrocytic end-feet from affected blood vessels, causing hypoxia of nervous system tissue. Astrocytes of rats and sheep affected by ETX show overexpression of aquaporin-4, a membrane channel protein that is believed to help remove water from affected perivascular spaces in an attempt to resolve the perivascular edema. Amyloid precursor protein, an early astrocyte damage indicator, is also observed in the brains of affected sheep. These results show that ETX activation in vivo seems to be more complex than previously thought and this toxin acts on the brain, affecting vascular permeability, but also damaging neurons and other cells.

ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE RED DEER (CERVUS ELAPHUS) OVER A 13-MONTH PERIOD.

Deer are sensitive to clostridial diseases, and vaccination with clostridial toxoids is the method of choice to prevent these infections in ruminants. The purpose of this study was to evaluate the serologic responses in red deer (Cervus elaphus) over a 13-mo period after vaccination with a multivalent clostridial vaccine, containing an aluminium hydroxide adjuvant. Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay. Animals from group 1 (9 mo old; n = 6) were naïve and received an initial vaccination with a booster vaccine 4 wk apart and one annual booster. Animals from group 2 (21 mo old; n = 10) had been previously vaccinated 12 mo prior and received a first annual booster at the beginning of this study and a second annual booster 12 mo later. The multivalent clostridial vaccine induced a high antibody response that peaked after each injection and then slowly decreased with time. In group 1, a booster vaccine was required to obtain an initial high humoral response. The annual booster injection induced a strong, rapid, and consistent anamnestic response in both groups. The serologic responses persisted significantly over the baseline value for 9-12 mo in group 1, but more than 12 mo in group 2. It is unknown whether the measured humoral immune responses would have been protective as no challenge studies were performed. Further investigation is needed to determine the protective antibody titers to challenge and how long this immunity might persist after vaccination.