Β2 Toxin Research Articles

A new method for rapid, portable, low-cost detection of Clostridium perfringens β2 toxin in animal fecal using smartphone-based electrochemical immunosensor

Clostridium perfringens (Cp) β2 toxin poses a significant threat to the breeding industry because of its potential to cause enterotoxaemia in livestock. However, existing quantitative methods are very scarce. Thus, we developed a rapid, portable, low-cost quantitative detection method for Cp β2 toxin using a smartphone-based electrochemical immunosensor. Recombinant 32a-β2 toxin protein was successfully prepared as an antigen, as well as its monoclonal antibody. As an alternative to the traditional three-electrode system, a ternary nanocomposite-modified commercially integrated three-electrode chip was fabricated. A smartphone-based electrochemical immunosensor was constructed by combining a low-cost handheld potentiostat with a smartphone, and it was compared with a large-scale potentiostat. Under optimal conditions, the portable sensor for identifying Cp β2 strain expression displayed a linear relationship from 0.1 to 100 ng mL−1, a low detection limit of 16.45 pg mL−1, high sensitivity, a low cost, easy operation, outdoor rapid detection, and satisfactory practicability and reliability.

Establishment of PCR assays and genetic polymorphism analysis of genes encoding Clostridium perfringens β2 toxin from different sources

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β2 toxin (cpb2) and atypical-cpb2 (aty-cpb2), analyze the epidemiological characteristics and genetic polymorphism of the cpb2 of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb2 of 188 Clostridium perfringens strains were examined by PCR; the cpb2 sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb2-library based on 110 strains carrying the cpb2 were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb2 (con-cpb2) and aty-cpb2. Results: The specificity of PCR assay for the cpb2 and aty-cpb2 was verified. The PCR results for cpb2 amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb2, 94 types A strains carried aty-cpb2, 6 types A strains carried con-cpb2, and 7 types F strains carried aty-cpb2. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb2 toxin was developed, and the previous PCR method for detecting aty-cpb2 was improved. aty-cpb2 is the primary gene encoding of β2 toxin. There is a significant nucleotide sequence variance between the various cpb2 genotypes.

Molecular detection and characterization of Clostridium perfringens toxin genes causing necrotic enteritis in broiler chickens.

A total of 464 samples comprising of cloacal swabs from necrotic enteritis suspected live birds (191), intestinal scrapings from dead birds with symptoms of necrotic enteritis (91), and apparently healthy birds (182) were collected from selected districts of AP. The samples were subjected to multiplex PCR for simultaneous detection of α, β, and β2 toxin genes and uniplex PCR for the detection of NetB gene. Multiplex PCR screening of samples reveled α toxin gene positives from (cpa) 248/282 (87.94%) necrotic enteritis suspected and 40/182 (21.97%) apparently healthy samples. Among cpa positives 142/248 (57.25%) and 3/40 (7.5%) were positive for β2 toxin gene in necrotic enteritis suspected and apparently healthy birds respectively, indicating the involvement of C. perfringens type A, with minor pore forming toxin gene cpb2 in causing necrotic enteritis in poultry. None of the sample was positive for β toxin gene. The present research indicates C. perfringens type A along with β2 toxin gene was responsible for causing necrotic enteritis in broiler chickens in some parts of Andhra Pradesh in India. Phylogenetic relationship of amplified cpa and cpb2 amino acids sequences from present C. perfringens isolates were studied. The analysis reveals the sequence identity of cpb2 gene of the present isolates and variations at both nucleotide and amino acid level with the published sequences of cpb2 gene of C. perfringens isolates from different animal species of the USA, Iran, Netherlands, and Japan.

The prevalence of plasmid-coded cpe enterotoxin, β2 toxin, tpeL toxin, and tetracycline resistance in Clostridium perfringens strains isolated from different sources

Clostridium perfringens, an anaerobic, spore-forming bacterium, causes infections in humans and animals by producing several toxins encoded by genes found either on the chromosomes or on diverse plasmids. The plasmids may code for more than one toxin gene or antimicrobial-resistance gene. In this study, the prevalence of the β2, cpe and tpeL toxin genes and the tetA(P), tetB(P) and tetM tetracycline-resistance genes, in 56 strains of C. perfringens type A isolated from diseased domestic animals and 15 strains isolated from chickens, was compared with that in 74 strains isolated from other sources. The frequency of chromosome-associated cpe enterotoxin genes was higher in strains not isolated from diseased domestic animals; however, plasmid-associated cpe genes were found in strains from some animal sources more than others. Enterotoxin production was detected in some strains that had chromosomal or plasmid cpe genes, but not in all. The percentages of strains carrying β2 toxin genes among chicken, swine, human patient and soil isolates were higher than those among bovine, canine and food isolates. The incidence of the tpeL toxin gene was lower than that of the β2 gene. Phenotypic resistance to tetracycline was found in more than 50% of the porcine, bovine, and canine isolates, which carried a wide range of plasmids of 2–100 kb size, most of which had the tcpH clostridial transfer gene. PCR amplified tetA(P) and tetB(P) genes from most isolates from diseased animals. Some strains that carried <40 kb plasmids and had the tcpH gene also had one or more toxin genes or tetracycline-resistance gene. This study shows that the prevalence of plasmid-borne toxins and antimicrobial resistance genes varied among C. perfringens strains isolated from different sources. Plasmids of smaller size than those previously reported in strains of C. perfringens type A may also harbor toxin genes and antimicrobial-resistance genes.

Detection of Clostridium perfringens toxinotypes, enteropathogenic E. coli, rota and corona viruses in the intestine of neonatal goat kids by molecular techniques

The role of various pathogenic agents affecting gastro-intestinal tract of neonatal goat kids was studied. Intestinal tissue samples of 142 necropsied kids died due to enteritis, pneumo-enteritis, tapeworm’s infestations and septicemia were collected for detection of toxinotypes of Clostridium perfringens, enteropathogenic E. coli (EPEC), Group-A rotavirus (GARV) and bovine coronavirus (BCV). C. perfringens toxinotyping was done by multiplex PCR using primers for cpa, cpb, cpb2, etx and iap gene. Further, identification of EPEC was done using SYBR green based real-time PCR assay targeting bfpA gene. Detection of GARV and BCV was done by one-step RT-PCR (osRTPCR). The incidence of C. perfringens was 16.20% (47.83% toxinotype A and 52.17% toxinotype D), with b2- toxin present in 30.43% of the samples. Incidence of EPEC in 0-1 and 1–3 month diarrhoeic kids was 36.62% and 25.35%, respectively. For viral aetiology, 11.97% were positive for GARV and 9.86% positive for BCV. Mixed infection of C. perfringens and EPEC was seen in 12.68% cases, while it was 2.11% for C. perfringens + GARV and 1.40% for C. perfringens + BCV. Similarly, the mixed occurrence of EPEC+GARV was 10.56% and that of EPEC+BCV was 7.04%. The combined infection of EPEC+GARV+BCV was 0.70%. The results suggested that C. perfringens type A and type D are the common toxinotypes affecting neonatal goat kids, with β2 toxin being an additional potential virulence factor. SYBR green based real-time PCR assay can be used as quick lab-based technique for efficient screening of EPEC from enteritis affected kids.Group-A rotavirus and bovine coronavirus appeared to be instrumental in causing mixed infection enteritis in kids.

Prevalence, toxin gene profile, antibiotic resistance, and molecular characterization of Clostridium perfringens from diarrheic and non-diarrheic dogs in Korea.

Clostridium perfringens causes diarrhea and other diseases in animals and humans. We investigated the prevalence, toxin gene profiles, and antibiotic resistance of C. perfringens isolated from diarrheic dogs (DD) and non-diarrheic dogs (ND) in two animal hospitals in Seoul, Korea. Fecal samples were collected from clinically DD (n = 49) and ND (n = 34). C. perfringens was isolated from 31 of 49 DD (63.3%) and 21 of 34 ND dogs (61.8%). All C. perfringens strains were positive for the α toxin gene, but not for the β, ε, or ι toxin genes; therefore, all strains were identified as type A C. perfringens. All isolates were cpe-negative, whereas the β2 toxin gene was identified in 83.9% and 61.9% of isolates from DD and ND, respectively. Most isolates were susceptible to ampicillin (94%), chloramphenicol (92%), metronidazole (100%), moxifloxacin (96%), and imipenem (100%). However, 25.0% and 21.2% of isolates were resistant to tetracycline and clindamycin, respectively. Molecular subtyping of the isolated strains was performed by using pulsed-field gel electrophoresis. Fifty-two isolates were classified into 48 pulsotypes based on more than 90% similarity of banding patterns. No notable differences were observed among the isolates from DD and ND.

Prevalence of C. Botulinum and C. Perfringens Spores in Food Products Available on Polish Market.

IntroductionThe aim of this study was to evaluate the prevalence of Clostridium botulinum and Clostridium perfringens in food samples purchased from Polish producers.Material and MethodsThe analyses were performed on 260 food samples collected in Lublin and Subcarpathian regions: 56 of smoked meat, 21 of pork meat, 20 of dairy products, 26 of vegetable and fruit preserves, 40 of ready-to-eat meals, 27 of fish preserves, and 70 of honey collected directly from apiaries.ResultsC. botulinum strains were isolated from 2.3% (6/260) of samples and the isolates were classified as toxin types A (4/260) and B (2/260). C. perfringens strains were isolated from 14% (37/260) of samples. All the isolates were classified as toxin type A, 28 of them were able also to produce α toxin and 9 – β2 toxin.ConclusionOn the basis of the obtained results it could be suggested that risk assessment, especially regarding the entire honey harvesting process, should be provided in order to ensure the microbiological safety of the products to be consumed by infants and people with a weakened immune system.

Clostridium perfringens Type A Isolates of Animal Origin with Decreased Susceptibility to Metronidazole Show Extensive Genetic Diversity.

Metronidazole (MTZ) is a first-line drug for the treatment of human infections caused by anaerobic bacteria. Although most Clostridium perfringens isolates are susceptible to metronidazole, a number of strains with decreased susceptibility have been obtained in clinical and environmental surveys. However, little is known yet about the toxinotype and genetic diversity of metronidazole-resistant C. perfringens strains. We tested for susceptibility to metronidazole and assessed the toxigenic status and amplified fragment length polymorphism (AFLP) diversity of a collection of 80 C. perfringens isolates of animal origin. In vitro susceptibility testing by the standard agar dilution technique revealed that 22 isolates (27.5% of total) were susceptible to metronidazole, 54 (67.5%) intermediate susceptible, and four (5%) resistant. All isolates belonged to toxinotype A, and two, 41 and 17, of them yielded a positive PCR result for presence of the gene encoding for enterotoxin (cpe), the consensus form of β2 toxin (cpb2) and the atypical form of β2 toxin, respectively. AFLP fingerprinting classified isolates into 65 genotypes indicating a high molecular diversity among isolates in this study, and this diversity was reflected among isolates from each susceptibility category. In conclusion, the results of this study revealed extensive genetic diversity among C. perfringens type A isolates regardless of their susceptibility to metronidazole.

NetF-positive Clostridium perfringens in neonatal foal necrotising enteritis in Kentucky

TYPE A Clostridium perfringens has been associated with necrotising enteritis in 1–14-day-old foals in Kentucky (Donahue and Williams 2002, Timoney and others 2005) but the pathogenesis of the disease is...

Distribution of &lt;i&gt;Clostridium perfringens&lt;/i&gt; Isolates from Piglets in South Korea

ABSTRACTClostridium perfringens causes various digestive system disease symptoms in pigs. In the present study, 11 C. perfringens isolates were obtained from diarrheic piglets and 18 from healthy piglets. All of the C. perfringens isolates were shown to be type A using a multiplex PCR assay. The β2 toxin gene was detected in 27/29 C. perfringens isolates, i.e., 81% (9/11) of diarrheic piglets and 100% (18/18) of healthy piglets, and all of the genes had the same sequence. In conclusion, the β2 toxin gene of C. perfringens was distributed widely in Korean piglets regardless of the incidence of diarrhea, and there was no clear relationship with enteric disease. A pulsed-field gel electrophoresis analysis of DNA digested using SmaI demonstrated the non-clonal spread of C. perfringens isolates from piglets.

Prevalence and characterization of Clostridium perfringens from the faecal microbiota of elderly Irish subjects

The aim of this study was to investigate the diversity and composition of the intestinal microbiota of elderly subjects using a combination of culture-dependent techniques and 16S rRNA gene amplicon sequencing. The study was performed as part of the ELDERMET project, in which 368 faecal samples were assessed for viable numbers of Bifidobacterium spp., Lactobacillus spp. and Enterobacteriaceae on selective agar. However, the Bifidobacterium selective medium used also supported the growth of Clostridium perfringens, which appeared as distinct colonies and were subsequently characterized phenotypically and genotypically. All the isolates were confirmed as toxin biotype A producers. In addition, three isolates tested also had the genetic determinants for the β2 toxin. Of the 368 faecal samples assessed, C. perfringens was detected in 28 samples (7.6%). Moreover, C. perfringens was observed in samples from subjects in all the residence locations assessed but was most prevalent in subjects from long-stay residential care, with 71.4% of the samples (63.2% of the subjects) being from this residence location, and with a shedding level in excess of 10(6) c.f.u. (g faeces)(-1). Microbiota profiling revealed some significant compositional changes across both the family and genus taxonomic levels between the C. perfringens-positive and -negative datasets. Levels of culturable Bifidobacterium spp. and Lactobacillus spp. were significantly (P<0.05) lower in the C. perfringens-positive samples. Sequence-based methods also confirmed a significant difference in the Bifidobacterium spp. level (P<0.05) between both datasets. Taken together, these data suggest that a high viable count [>10(6) c.f.u. (g faeces)(-1)] of C. perfringens in stool samples may be indicative of a less healthy microbiota in the intestine of elderly people in long-stay residential care.

EL COMPLEJO ENTÉRICO NEONATAL EN ALPACAS ANDINAS.

Advances in research on the major causative agents of morbidity and mortality in newborn alpacas associated with enteric processes in southern Peru were reviewed. Microbiology and molecular analyses performed on intestinal samples from enterotoxemia fatalities confirmed the predominance of C. perfringens type A carrying only the gene coding for the major α exotoxin and identifiying for the first time the presence of the novel β2 toxin gene. In vitro studies have yielded three profiles for phospholipase activity (high, medium and low) with biological activity when high and medium strains were inoculated intraintestinally in mice and rabbits, but did not induce intestinal pathology in an alpaca cria. A detailed histopathological investigation has reveled that within necrotizing hemorrhagic enteritis Clostridium coexist with massive presence of Eimeria macusaniensis suggesting that primary parasite tissue destruction may well predispose overgrowth of clostridium and toxin production, triggering enteric fatalities. Additionally, studies on diarrheas intestinal swabs and/or intestinal contents identified Escherichia coli pathogenic strains (enteropathogenic and enterohemorrhagic). The immunofluorescent direct test and PCR revealed the presence of Giardia intestinalis coexisting with mostly enteropathogenic and enterohemorrhagic E. coli strains, as well as with a virus similar to bovine coronavirus. These microbes are potentially diarrheagenic pathogens and a possible infection source for Andean people.

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

BackgroundClostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status.ResultsToxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, β2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good.ConclusionsClostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.

Diffusion of Clostridium perfringens NetB positive strains in healthy and diseased chickens

For over 30 years α toxin was considered the key virulence factor responsible for the appearance of necrotic enteritis (NE) in chickens but, recently, a new toxin related to the occurrence of NE, called NetB, has been described. The aim of this work was to evaluate the CP toxin-type and the NetB gene presence in strains collected from chickens affected or not by enteric diseases. 107 strains were tested: 30 isolated from chickens affected by NE, 54 from subjects affected by other enteric pathologies and 22 from healthy animals. All strains resulted toxin-type A and 26.17% of these was positive also for β2 toxin gene. No strains were positive for cpe gene. 27% (29/107) of CP was NetB positive and 93% (27/29) of these was isolated from birds affected by intestinal disorders. 16 NetB positive strains were obtained from chickens affected by NE (16/30), 9 from animals affected by other intestinal disorders (9/54) and 4 from healthy animals (4/22). A significant difference between the number of NetB positive strains isolated from animals affected by NE and healthy chickens has been observed (P=0.014). However, the finding that the 17.4% of strains isolated from healthy chickens was also positive for NetB, confirm that other virulence factors could play an important role on NE appearance.

Enterotoxemia Associated with Beta2 Toxin–Producing Clostridium Perfringens Type A in Two Asiatic Black Bears (Selenarctos Thibetanus)

Beta2 (beta2) toxin-producing Clostridium perfringens type A strains were found to be associated with necrotic and hemorrhagic intestinal lesions in 2 Asiatic black bears (Selenarctos thibetanus) that died suddenly. Ten isolates were obtained from the liver, lungs, heart, and small and large intestine of the animals and were examined by multiplex polymerase chain reaction for the genes encoding the 4 lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types as well as for the genes encoding enterotoxin and the novel beta2-toxin for subclassification. In addition, the cpb2 sequence of the 10 isolates was different from the published sequence of cpb2 of pig type C isolate CWC245, whereas it was highly similar to the cpb2 sequence of the C. perfringens type A strain 13. This finding suggests the existence of 2 cpb2 subtypes. This is the first report of enterotoxemia associated with the presence of C. perfringens producing beta2-toxin in the tissues and intestinal content of Asiatic black bears.

Clostridium perfringens toxin types from wild-caught Atlantic cod (Gadus morhua L.), determined by PCR and ELISA.

Ninety-five fecal samples from Atlantic cod (Gadus morhua L.), caught along the northern Norwegian coast, were examined bacteriologically for occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. In addition, a commercial enzyme-linked immunosorbent assay (ELISA) kit for detection of C. perfringens alpha, beta, and epsilon toxin was used. Clostridium perfringens could be isolated in 37 fecal samples (38.9%) from cod. All isolates were C. perfringens toxin type A (alpha toxin positive) as determined by PCR and also ELISA. In addition, in isolates from two cod (2.1%) the gene encoding for beta2 toxin was found (A, beta2) by PCR. Genes encoding for beta, epsilon, and iota toxins and enterotoxin were not found. This is the first detection of C. perfringens alpha and beta2 toxin in cod and of beta2 toxin in fish in general. The origin of this bacterium in cod is discussed.

A role for the Clostridium perfringens β2 toxin in bovine enterotoxaemia?

Non-enterotoxigenic type A Clostridium perfringens are associated with bovine enterotoxaemia, but the α toxin is not regarded as responsible for the production of typical lesions of necrotic and haemorrhagic enteritis. The purpose of this study was to investigate the putative role of the more recently described β2 toxin. Seven hundred and fourteen non-enterotoxigenic type A C. perfringens isolated from 133 calves with lesions of enterotoxaemia and high clostridial cell counts (study population) and 386 isolated from a control population of 87 calves were tested by a colony hybridisation assay for the β2 toxin. Two hundred and eighteen (31%) C. perfringens isolated from 83 calves (62%) of the study population and 113 (29%) C. perfringens isolated from 51 calves (59%) of the control population tested positive with the β2 probe. Pure and mixed cultures of four C. perfringens (one α+β2+, one α+enterotoxin+ and two α+) were tested in the ligated loop assay in one calf. Macroscopic haemorrhages of the intestinal wall, necrosis and haemorrhages of the intestinal content, and microscopic lesions of necrosis and polymorphonuclear and mononuclear cell infiltration of the intestinal villi were more pronounced in loops inoculated with the α and β2-toxigenic C. perfringens isolate. These results suggest in vivo synergistic role of the α and β2 toxins in the production of necrotic and haemorrhagic lesions of the small intestine in cases of bovine enterotoxaemia. However, isolation of β2-toxigenic C. perfringens does not confirm the clinical diagnosis of bovine enterotoxaemia and a clostridial cell counts must still be performed.

Population-based study of fecal shedding of Clostridium perfringens in broodmares and foals.

To determine the percentage of broodmares and foals that shed Clostridium perfringens in their feces and classify the genotypes of those isolates. Prospective cross-sectional study. 128 broodmares and their foals on 6 equine premises. Anaerobic and aerobic bacteriologic cultures were performed on feces collected 3 times from broodmares and foals. All isolates of C. perfringens were genotyped. Clostridium perfringens was isolated from the feces of 90% of 3-day-old foals and 64% of foals at 8 to 12 hours of age. A lower percentage of broodmares and 1- to 2-month-old foals shed C. perfringens in their feces, compared with neonatal foals. Among samples with positive results, C. perfringens type A was the most common genotype identified (85%); C. perfringens type A with the beta2 toxin gene was identified in 12% of samples, C. perfringens type A with the enterotoxin gene was identified in 2.1% of samples, and C. perfringens type C was identified in < 1% of samples. Clostridium perfringens was identified from the feces of all but 6 foals by 3 days of age and is likely part of the normal microflora of neonatal foals. Most isolates from broodmares and foals are C. perfringens type A; thus, the clinical relevance of culture results alone is questionable. Clostridium perfringens type C, which has been associated with neonatal enterocolitis, is rarely found in the feces of horses.

Detection of the β2 toxin gene of Clostridium perfringens in diarrhoeic piglets in The Netherlands and Switzerland

The two studies presented here were done to determine the prevalence of the α, β, ϵ and enterotoxin genes and the novel β2 toxin gene of Clostridium perfringens in neonatal or pre-weaned piglets with diarrhoea or necrotic enteritis. All C. perfringens isolates were positive for the α and negative for the ϵ and enterotoxin gene, implying that only non-enterotoxigenic type A and C strains were detected. The most important findings were the relatively high prevalence of the β2 toxin gene in isolates from diarrhoeic piglets in both studies, and, in one of the two studies, absence of strains with only the α and β toxin gene. These data are supportive for the suggestion of a causal relationship of β2 toxin-producing strains with digestive tract diseases in piglets.