Clostridium Perfringens Toxin Research Articles

Plasma proteome signature of canine acute haemorrhagic diarrhoea syndrome (AHDS).

Acute haemorrhagic diarrhoea is a common complaint in dogs. In addition to causes like intestinal parasites, dietary indiscretion, intestinal foreign bodies, canine parvovirus infection, or hypoadrenocorticism, acute haemorrhagic diarrhoea syndrome (AHDS) is an important and sometimes life-threatening differential diagnosis. There is some evidence supporting the link between Clostridium perfringens toxins and AHDS. These toxins may be partially responsible for the epithelial cell injury, but the pathogenesis of AHDS is still not fully understood. Recent studies have suggested that severe damage to the intestinal mucosa and associated barrier dysfunction can trigger chronic gastrointestinal illnesses. Besides bloodwork and classical markers for AHDS such as protein loss and intestinal bacterial dysbiosis, we focused mainly on the plasma-proteome to identify systemic pathological alterations during this disease and searched for potential biomarkers to improve the diagnosis. To accomplish the goals, we used liquid chromatography-mass spectrometry. We compared the proteomic profiles of 20 dogs with AHDS to 20 age-, breed-, and sex-matched control dogs. All dogs were examined, and several blood work parameters were determined and compared, including plasma biochemistry and cell counts. We identified and quantified (relative quantification) 207 plasmatic proteins, from which dozens showed significantly altered levels in AHDS. Serpina3, Lipopolysaccharide-binding protein, several Ig-like domain-containing proteins, Glyceraldehyde-3-phosphate dehydrogenase and Serum amyloid A were more abundant in plasma from AHDS affected dogs. In contrast, other proteins such as Paraoxonase, Selenoprotein, Amine oxidases, and Apolipoprotein C-IV were significantly less abundant. Many of the identified and quantified proteins are known to be associated with inflammation. Other proteins like Serpina3 and RPLP1 have a relevant role in oncogenesis. Some proteins and their roles have not yet been described in dogs with diarrhoea. Our study opens new avenues that could contribute to the understanding of the aetiology and pathophysiology of AHDS.

Co-administration of the prebiotic 1-kestose and the paraprobiotic Lactiplantibacillus plantarum FM8 in magellanic penguins promotes the activity of intestinal Lactobacillaceae and reduces the plc gene levels encoding Clostridium perfringens toxin.

Despite the well-known potential health benefits of prebiotics and non-viable probiotics (paraprobiotics) in various animal species, research regarding their use in penguins is scarce. Our study aimed to investigate the impact of a combined administration of prebiotics and paraprobiotics (referred to here as "parasynbiotics") on the gut microbiome and overall health of Magellanic penguins (Spheniscus magellanicus). The parasynbiotics consisted of 1-kestose, which is a fructooligosaccharide comprising sucrose and fructose, and heat-killed Lactiplantibacillus plantarum FM8, isolated from pickled vegetables. It was administered to eight penguins aged <3 years (Young-group) and nine penguins aged >17 years (Adult-group) for 8 weeks. Results from 16S rRNA sequencing revealed that compared to baseline, parasynbiotic administration significantly decreased the relative abundance of intestinal Clostridiaceae_222000 in both groups and significantly increased that of Lactobacillaceae in the Young-group. Quantitative real-time polymerase chain reaction revealed a significant decrease in the plc gene levels encoding alpha-toxin of Clostridium perfringens in the Young-group after parasynbiotic administration (P=0.0078). In the Young-group, parasynbiotic administration significantly increased the plasma levels of total alpha-globulin (P=0.0234), which is associated with inflammatory responses. Furthermore, exposure of dendritic cells to heat-killed L. plantarum FM8 promoted the secretion of interleukin 10, a major anti-inflammatory cytokine. Overall, parasynbiotic administration enhanced the activity of gut Lactobacillaceae, decreased the levels of C. perfringens and its toxin encoding plc gene, and reduced inflammatory response in penguins. These results provide novel insights into the potential benefits of parasynbiotics for improving penguin health.

Ensuring veterinary well-being and productivity of pigs with an immunotropic drug

The purpose of this work is to realize the productive qualities of young pigs by increasing the effectiveness of specific prevention of infectious diseases. The object of the study was 45 pigs of the Landrace breed from 14 to 171 days of age, divided according to the principle of pairs of analogues into 3 groups of 15 heads. The animals of the 1st experimental group were injected with the immunotropic drug PigStim-V in a dose of 1.0 ml per head twice at the age of 14 and 21 days in order to stimulate nonspecific resistance of the body and increase the effectiveness of specific prevention of infectious diseases intramuscularly at the age of 14 and 21 days. Moreover, the second injection was carried out simultaneously with immunization against type 2 porcine circovirus infection. The tested immunotropic drug PigStim-V was administered to animals of the 2nd experimental group once, at a dose of 1.0 ml per head at the age of 21 days simultaneously with a commercial vaccine against porcine circovirus. Animals of the 3rd group served as biological control. The results of the study confirm the effectiveness of the use of the immunotropic drug PigStim-V as an adjuvant in the vaccination of pigs. Both single and double intramuscular injection of PigStim-V increases the titer of specific antibodies against porcine circovirus infection and against α, β and ε toxins of the microorganism Clostridium perfringens, prevents and increases the effectiveness of therapy of diseases of non-infectious etiology, stimulates growth. A more pronounced positive effect is observed with the double administration of an immunotropic drug.

Progress in research of Clostridium perfringens toxin

Clostridium perfringens can produce many kinds of toxins and hydrolase, causing gas gangrene, enteritis and enterotoxemia in both human and animals. It is known that C. perfringens can produce more than 20 toxins and hydrolases. The different toxin types are associated with specific disease types. At present, molecular toxin-typing method by PCR has replaced the traditional serological typing method. In this study, we systematically summarize the types, basic characteristics, pathogenic mechanism and the relationship with disease of C. perfringens toxins to provide evidence for the establishment of rapid detection method, immune antigen screening, antibody preparation and research of related pathogenic mechanism.

Lactobacillus casei displaying Clostridium perfringens NetB antigen protects chickens against necrotic enteritis.

Necrotic enteritis is a serious economical disease of poultry caused by Clostridium perfringens. NetB toxin of Clostridium perfringens is considered the causative agent of necrotic enteritis. Following the withdrawal of in-feed antibiotic growth promoters, there has been an urgent need to develop alternative approaches such as vaccination. Currently, there are no commercially available vaccines to control necrotic enteritis especially in broiler chickens as the target population. In the present study, we constructed a recombinant Lactobacillus casei strain expressing NetB protein of C. perfringens on the cell surface and used this probiotic-based vaccine strain to immunize broiler chickens orally against experimental induction of necrotic enteritis. The birds immunized with the oral vaccine strain were significantly protected against necrotic enteritis challenge and developed strong serum anti-NetB antibody responses to NetB protein. Furthermore, the immunized birds showed higher body weight gains during the challenge experiment compared with control birds. This study showed, for the first time, that a probiotic-based vector vaccine could be a promising vaccine candidate to provide protection against necrotic enteritis in broiler chickens. KEYPOINTS: • The probiotic L. casei carrying pT1NX-netB plasmid displayed NetB antigen on the cell surface. • The LC-NetB vaccine strain induced high anti-toxin antibody response in broiler chickens. • The LC-NetB vector vaccine provided significant protection against experimental NE challenge.

Short and Medium Chain Fatty Acids and Their Derivatives as a Natural Strategy in the Control of Necrotic Enteritis and Microbial Homeostasis in Broiler Chickens.

The use of antibiotic growth promoters (AGPs) has historically been the most important prophylactic strategy for the control of Necrotic Enteritis (NE) caused by some Clostridium perfringens toxin types in poultry. During the last five decades, AGPs have also been supplemented in feed to improve body weight gain and feed efficiency as well as to modulate the microbiome (consisting of microbes and their genes both beneficial and potentially harmful) and reduce enteric pathogens, among other benefits. New regulatory requirements and consumer preferences have led to strong interest in natural alternatives to the AGPs for the prevention and control of illnesses caused by enteric pathogens. This interest is not just focused on the direct removal or inhibition of the causative microorganisms but also the improvement of intestinal health and homeostasis using a range of feed additives. A group of promising feed additives is short- and medium-chain fatty acids (SCFA and MCFA, respectively) and their derivatives. The use of SCFA and MCFA, including butyric, caproic, caprylic, capric, and lauric acids, has shown strong effects against NE in broilers both at experimental and commercial levels. These fatty acids also benefit intestinal health integrity and homeostasis. Other effects have also been documented, including increases in intestinal angiogenesis and gene expression of tight junctions. Chemical modifications to improve stability and point of release in the intestine have been shown to improve the efficacy of SCFA and MCFA and their derivatives. The aim of this review is to give an overview of SCFA, MCFA and their derivatives, as an alternative to replace AGPs to control the incidence and severity of NE in poultry.

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.

Clostridium Perfringens Toxin Types Associated with Meat: Review in Iran

Clostridium Perfringens Toxin Types Associated with Meat: Review in Iran

Identifying the Basis for VirS/VirR Two-Component Regulatory System Control of Clostridium perfringens Beta-Toxin Production.

Clostridium perfringens toxin production is often regulated by the Agr-like quorum sensing (QS) system signaling the VirS/VirR two-component regulatory system (TCRS), which consists of the VirS membrane sensor histidine kinase and the VirR response regulator. VirS/VirR is known to directly control expression of some genes by binding to a DNA binding motif consisting of two VirR boxes located within 500 bp of the target gene start codon. Alternatively, the VirS/VirR system can indirectly regulate production levels of other proteins by increasing expression of a small regulatory RNA, VR-RNA. Previous studies demonstrated that C. perfringens beta-toxin (CPB) production by C. perfringens type B and C strains is positively regulated by both the Agr-like QS and the VirS/VirR TCRS, but the mechanism has been unclear. The current study first inactivated the vrr gene encoding VR-RNA to show that VirS/VirR regulation of cpb expression does not involve VR-RNA. Subsequently, bioinformatic analyses identified a potential VirR binding motif, along with a predicted strong promoter, ∼1.4 kb upstream of the cpb open reading frame (ORF). Two insertion sequences were present between this VirR binding motif/promoter region and the cpb ORF. PCR screening of a collection of strains carrying cpb showed that the presence and sequence of this VirR binding motif/promoter is highly conserved among CPB-producing strains. Reverse transcription-PCR (RT-PCR) and a GusA reporter assay showed this VirR binding motif is important for regulating CPB production. These findings indicate that VirS/VirR directly regulates cpb expression via VirS binding to a VirR binding motif located unusually distant from the cpb start codon. IMPORTANCE Clostridium perfringens beta-toxin (CPB) is only produced by type B and C strains. Production of CPB is essential for the pathogenesis of type C-associated infections, which include hemorrhagic necrotizing enteritis and enterotoxemia in both humans and animals. In addition, CPB can synergize with other toxins during C. perfringens gastrointestinal diseases. CPB toxin production is cooperatively regulated by the Agr-like quorum sensing (QS) system and the VirS/VirR two-component regulatory system. This study now reports that the VirS/VirR regulatory cascade directly controls expression of the cpb gene via a process involving a VirR box binding motif located unusually far (∼1.4 kb) upstream of the cpb ORF. This study provides a better understanding of the regulatory mechanisms for CPB production by the VirS/VirR regulatory cascade.

The MAL Protein, an Integral Component of Specialized Membranes, in Normal Cells and Cancer.

The MAL gene encodes a 17-kDa protein containing four putative transmembrane segments whose expression is restricted to human T cells, polarized epithelial cells and myelin-forming cells. The MAL protein has two unusual biochemical features. First, it has lipid-like properties that qualify it as a member of the group of proteolipid proteins. Second, it partitions selectively into detergent-insoluble membranes, which are known to be enriched in condensed cell membranes, consistent with MAL being distributed in highly ordered membranes in the cell. Since its original description more than thirty years ago, a large body of evidence has accumulated supporting a role of MAL in specialized membranes in all the cell types in which it is expressed. Here, we review the structure, expression and biochemical characteristics of MAL, and discuss the association of MAL with raft membranes and the function of MAL in polarized epithelial cells, T lymphocytes, and myelin-forming cells. The evidence that MAL is a putative receptor of the epsilon toxin of Clostridium perfringens, the expression of MAL in lymphomas, the hypermethylation of the MAL gene and subsequent loss of MAL expression in carcinomas are also presented. We propose a model of MAL as the organizer of specialized condensed membranes to make them functional, discuss the role of MAL as a tumor suppressor in carcinomas, consider its potential use as a cancer biomarker, and summarize the directions for future research.

Effects of vaccination against brucellosis and clostridia on the intake, performance, feeding behavior, blood parameters, and immune responses of dairy heifers calves.

The aim of this study was to identify possible effects of different vaccination strategies (concomitantly or not) against brucellosis and clostridia on intake, performance, feeding behavior, blood parameters, and immune responses of dairy heifers calves. Fifty heifers calves were enrolled [38 Gyr (Zebu, Bos taurus indicus) and 12 5/8 Holstein × Gyr]. At 120 d of age, animals were randomly distributed among 3 groups: B (n = 18), vaccinated against brucellosis; C (n = 14), vaccinated against clostridia and CB (n = 18), vaccinated concomitantly for both. Rectal and thermographic temperatures were evaluated on days -1, 0, 1, 2, 3, 5, 7,10, 14, and 28 relatives to the vaccination day. Feed and water intake, body weight (BW), and feeding behavior were monitored daily by an electronic feeding system. Blood was sampled on days 0, 3, 7, 14, and 28, relative to the vaccination day for determination of glucose and β -hydroxybutyrate (BHBA) concentrations. Blood sampled on day 0 (prevaccination) and on days 28 and 42 were used to evaluate the immune response against Brucella abortus and clostridia. There was an increase in rectal temperature between the first and the third day postvaccination in the 3 groups. The thermography revealed an increase of local temperature for 7 d on groups B and CB. Group C had increased local temperature for a longer period, lasting for up to 14 d. Dry mater intake was reduced for groups B and CB, but no alteration was observed for group C. No alterations regarding initial BW, final BW, average daily weight gain, and feed efficiency were observed. No differences were observed for the 3 vaccination groups for blood parameters throughout the evaluation period. The concomitant vaccination against brucellosis and clostridia led to lower neutralizing antibody titers against epsilon toxin of Clostridium perfringens and botulinum toxin type C of C. botulinum (C > CB > B). When cellular proliferation assay and serological tests to B. abortus were evaluated, no differences were observed between groups B and CB. The present results indicate that the concomitant vaccination against brucellosis and clostridia has no relevant impact on the intake, performance, and feeding behavior of dairy calves. However, the concomitant vaccination of vaccines against these 2 pathogens impacts animal immunity against clostridial infections.

Current notions about etiopathogenic and genetics specific features of &lt;i&gt;Сlostridium perfringens&lt;/i&gt; toxins

The review presents modern data on the genetics and etiopathogenetic features of Clostridium perfringens toxins, including the role of Clostridium perfringens enterotoxin, in the development of food poisoning and a number of intestinal diseases in humans, animals and birds.

Sequencing of Clostridium perfringens toxin genes (cpa, etx, iap) from Iraqi hospitals and detection by PCR of the genes encoding resistance to metronidazole, tetracycline, and clindamycin

PurposeThis cross sectional study was designed to investigate the molecular detection of C. perfringens toxins (alpha, epsilon, iota) and antibiotics resistance genes, as well as sequencing of their toxin genes. MethodsDifferent wound swabs were taken from 140 patients. PCR was applied for detection of clostridial toxins; alpha toxin (cpa) gene, epsilon toxin (etx) gene, and iota toxin (iap) gene. Metronidazole nim gene, tetracycline resistance genes; (tetQ, tetM, tetB, and tetW) and clindamycin resistance genes erm (A,B), were used for genotypic detection of antibiotic resistance. ResultsOut of 140 clinical samples collected, 7 isolates were detected using specific primer 16S–23S intergenic rRNA spacer gene of C. perfringens. Results showed presence of alpha toxin (cpa) genes in all clostridial isolates, Epsilon toxin (etx) genes in 2/7(28.4%), and iota toxin (iap) genes in 2/7 (28.4%). Results of antibiotic resistance genes showed that all isolates were not able to produce nim, tet W and Q, tet B, erm (A), erm (B) genes except for tetM. Gene sequence analysis for cpa gene showed that there were 3 mutations in the sample of this gene, the results of etx gene when had been sent 2 samples from this gene. The first sample showed that there was one mutation and the results of iap gene showed that there were no mutation in 2 samples of the iap gene. The samples had been showed identical 100%. ConclusionThe DNA sequence analysis of bacterial genome revealed several important feature including that may allow to confirm new the presence of toxin and to identify new or mutant toxins that may be missed by diagnostic PCR specifically target toxin encoding genes.

Harnessing an Integrative In Silico Approach to Engage Highly Immunogenic Peptides in an Antigen Design Against Epsilon Toxin (ETX) of Clostridium perfringens

Epsilon toxin (ETX) is one of four lethal toxins of Clostridium perfringens produced by types B and D of the pathogen. This pore-forming toxin is one of the most potent bioterrorism agents with economic importance. Although an effective vaccine and an equine antitoxin are available to protect livestock against ETX, no approved vaccine or antitoxin is available for humans. In the current study, an integrative, simple, fast, and reliable approach is availed to design a safe and minimized construct based on preexisting experimental linear B cell epitopes. This guideline is designed based on the surface accessibility, flexibility, hydrophilicity, content of beta-turn structure, and antigenicity of potential epitopes. Experimental linear B-cell epitopes were analyzed with respect to antigenicity. Two antigenic regions were introduced based on the distribution of epitopes. Moreover, three constructs were designed based on top ranking epitopes. These constructs were evaluated for their antigenicity, surface accessibility, flexibility, hydrophilicity, and beta-turn content and were compared to the ETX sequence. The 114-aa construct with an antigenicity score of 0.8592 was the most stable, antigenic, hydrophilic, flexible, and surface accessible antigen within the ETX sequence, the selected regions, and the designed constructs. Although the performed in silico analyses revealed that the designed construct could serve as a safe antigen triggering highly reactive and neutralizing anti-ETX antibodies, it should be verified by experimental assays in future studies.

Varying starch to fat ratios in pelleted diets: II. Effects on intestinal histomorphometry, Clostridium perfringens and short-chain fatty acids in Eimeria-challenged broiler chickens

ABSTRACT 1. The hypothesis behind the study was that a high dietary starch level (HS) would lead to impaired gut health compared to a low-starch diet (LS) in Eimeria-challenged broilers. The effects of two diets with different starch to fat ratios on intestinal histomorphometry, Clostridium perfringens counts and toxin profile, necrotic enteritis prevalence and abundance of short-chain fatty acids (SCFAs) were examined. 2. A total of 1,920 one-day-old Ross 308 broiler chickens were fed one of two isocaloric diets formulated either with high (32:1) or low (2:1) starch to fat ratios from d 10 to 29 of age. Each treatment group had 12 pen replicates containing 80 broilers each. On d 17, the chickens were challenged with Eimeria vaccine strains. Samples were collected on d 16, 21–23 and 29. 3. Whereas villus length increased gradually throughout the study in the HS group, a peak level was reached on d 21–23 in the LS group. On d 29, the HS group had significantly longer villi than the LS group. 4. Caecal SCFA concentrations were higher in the HS group compared to the LS group on d 16. In both groups, the SCFA level peaked on d 21–23, with the most pronounced increase seen in the LS group. 5. The C. perfringens netB:cpa ratio increased from d 16 to 29 in the HS group. C. perfringens counts and necrotic enteritis prevalence were similar between the two groups. 6. Diet affected the dynamics of small intestinal villus length and caecal SCFA abundance. These findings suggest that structural remodelling of the small intestine is an adaptation to different dietary starch levels, and that caecal SCFA abundance is associated with the availability of substrate for the microbiota in the posterior intestinal segments. Chickens adapted to higher levels of dietary starch might be more robust against Eimeria infections due to increased mucosal surface area. Studies with other dietary starch sources are required to clarify the impact of dietary starch levels on intestinal health in Eimeria–challenged broilers.

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.

Developing an experimental necrotic enteritis model in turkeys - the impact of Clostridium perfringens, Eimeria meleagrimitis and host age on frequency of severe intestinal lesions

BackgroundNecrotic enteritis is a significant problem to the poultry industry globally and, in Norway up to 30% of Norwegian turkey grow-outs can be affected. However, despite an awareness that differences exist between necrotic enteritis in chickens and turkeys, little information exists concerning the pathogenesis, immunity, microbiota or experimental reproduction of necrotic enteritis in turkeys. In particular, it is important to determine the appearance of the gross lesions, the age dependency of the disease and the role of netB toxin of Clostridium perfringens. To this end, we report our findings in developing an in vivo experimental model of necrotic enteritis in turkeys.ResultsA four tier (0–3) scoring system with clearly defined degrees of severity of macroscopic intestinal lesions was developed, based on 2312 photographic images of opened intestines from 810 B.U.T. 10 or B.U.T. Premium turkeys examined in nine experiments. Loss of macroscopically recognizable villi in the anterior small intestine was established as the defining lesion qualifying for a score 3 (severe intestinal lesions). The developed scoring system was used to identify important factors in promoting high frequencies of turkeys with severe lesions: a combined Eimeria meleagrimitis and Clostridium perfringens challenge, challenge at five rather than 3 weeks of age, the use of an Eimeria meleagrimitis dose level of at least 5000 oocysts per bird and finally, examination of the intestines of 5-week-old turkeys at 125 to 145 h after Eimeria meleagrimitis inoculation. Numbers of oocysts excreted were not influenced by Clostridium perfringens inoculation or turkey age. Among three different lesion score outcomes tested, frequency of severe lesions proved superior in discriminating between impact of four combinations of Clostridium perfringens inoculation and turkey age at challenge.ConclusionsThis study provides details for the successful establishment of an in vivo model of necrotic enteritis in turkeys.

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

BackgroundClostridium 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.ResultsIn 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.ConclusionsThe 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.

The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance

Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.

Clostridium perfringens Foodborne Outbreak during an Athletic Event in Northern Greece, June 2019.

Background: Food safety is a major public health consideration during athletic events. On 27 June 2019, the Hellenic National Public Health Organization was notified of a cluster of gastroenteritis cases among athletes of four of the 47 teams participating at the Panhellenic Handball Championship for children. Methods: A retrospective cohort study among the members of the four teams was performed. The local public health authority visited the restaurants where common meals took place, amassed information on the preparation of meals, and collected samples of leftovers. Stool samples were tested for Salmonella spp. and Shigella spp. Results: Consumption of minced beef had a statistically significant association with disease occurrence [RR:8.29 (95%CI 1,31-52,7)]. Samples of meat were found positive for Clostridium perfringens. It was documented that the meat was not stored and re-heated as indicated. Stool samples were negative for Salmonella spp. and Shigella spp. and were not tested for the Clostridium perfringens toxin. Conclusion: Specific standards should be kept to prevent outbreaks during athletic events. This was the first time that a foodborne outbreak due to Clostridium perfringens was investigated in the country. Laboratory investigation for toxins should be enhanced, especially in foodborne outbreaks where clinical manifestations of cases are found to be compatible with infection caused by a toxin.