Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Affinity, protease susceptibility, thermostability, and production levels of parent and mutant Nb clones

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Mutagenesis, Sequencing

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Cloning, Plasmid Preparation, Marker, Isolation

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Llama immunization scheme (a) and immune response of SNL‐133 (b and d) and SNL‐134 (c and e) to directly coated α toxin (b and c) and NetB (d and e). Two llamas, SNL133 and SNL134, were immunized with NetB and α toxin toxoids on Day 0, Day 14, Day 28, Day 35, Day 57, and Day 71, and sera were collected at different time points and analyzed for an immune response using ELISA. Representative results are shown from three independent experiments

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Enzyme-linked Immunosorbent Assay

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Binding of the periplasmic fractions of the master plate EAT‐1 to α toxin (a) and binding of the periplasmic fractions of the master plate ENB‐1 to NetB (b). The binding strength is indicated by absorbance at 490 nm

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Binding Assay

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: SDS‐PAGE analysis of the purified VHHs and their dose response binding to NetB and α toxin. (a) Coomassie‐stained SDS‐PAGE showing analysis of purified VHH selected on NetB (1–5) and α toxin (6–12). R, 1 µg reference VHH; L, Prestained protein ladder (PageRuler, ThermoFisher); 1, ENB‐1A4; 2, ENB‐1F4; 3, ENB‐1B9; 4, ENB‐1F10; 5, ENB‐1D11; 6, EAT‐1A2; 7, EAT‐1F2; 8, EAT‐1G4; 9, EAT‐1F3; 10, EAT‐1D7; 11, EAT‐1A3; and 12, EAT‐1C8. (b) and (c) Dose response binding of the selected VHHs to α toxin (b) or NetB (c). Representative results are shown from three independent experiments

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: SDS Page, Purification, Binding Assay, Staining

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Neutralization of α toxin lecithinase and NetB hemolytic activity by VHH clones. (a, b) A twofold dilution series of VHH antibodies, starting from 5 µM concentration was preincubated with either (a) recombinant α toxin or (b) commercial α toxin, after which egg yolk solution was added. Serum derived from calves immunized with recombinant α toxin was used as a control (control serum). Egg yolk solution incubated with α toxin without VHH, or serum was used to calculate 100% activity. (c) A twofold dilution series of VHH, starting from 5 µM concentration, was preincubated with recombinant NetB (in a total volume of 2 µl), after which 1% chicken erythrocytes was added. Serum derived from rabbits immunized with recombinant NetB was used as a control (control serum). The optical density of 100% hemolysis (mean OD 570 = 0.37, indicated by solid line) was obtained by diluting the chicken erythrocytes in distilled water. As a control, chicken erythrocytes incubated with NetB, but without VHH or serum was used (mean OD 570 = 0.36, indicated by dotted line). This resulted in 100% hemolysis. A phosphate‐buffered saline control (1% chicken erythrocytes with no NetB or Nbs) resulted in a mean OD 570 of 0.03. Representative results are shown from three independent experiments. VHH, Variable domain of the Heavy chain of Heavy chain

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Neutralization, Activity Assay, Clone Assay, Concentration Assay, Recombinant, Derivative Assay, Control, Incubation, Saline

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Optimization of VHH clones for improved affinity, production, and stability. (a) Structure of ENB‐ID11 predicted based on homology modeling. The (b, c) Affinity of VHH clones to α toxin (EAT clones) and NetB (ENB clones). (d – g) SDS‐PAGE gel of stable VHH control (d), EGFR Q44C (protease susceptible control) (e), EAT‐1F2 (f), and ENB‐1D11_R56H (g) incubated with immobilized trypsin for different time points: 0, 15, 30, 45, 60, 90, and 120 min at 37°C. Representative results are shown from three independent experiments. VHH, Variable domain of the Heavy chain of Heavy chain

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Clone Assay, SDS Page, Control, Incubation

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Expression cassette and genetic manipulation toolkit used for integration of expression cassettes into L. reuteri genomes. (a) Expression cassette showing the cwlS secretion signal sequence, 5′ anchor sequence, optimized ENB‐1D11_R56H (anti‐NetB Nb), cwlS 3′ anchor, and cwlS terminator and flanking regions. (b) Schematic diagram of the suicide vector used for the integration of expression cassette and pyrE truncation (truncated pyrE is shown in the solid green block with no arrow), and the integration site in the L. reuteri genome. (c) Schematic diagram of the integration vector used for correcting pyrE (wildtype pyrE is shown in the solid green block with an arrow) and the integration site in the L. reuteri genome. (d) Agarose gel showing the PCR confirmation of the integration of the expression cassette and pyrE correction for EAT‐1G4 (anti‐α toxin Nb) and ENB‐1D11_R56H (anti‐NetB Nb). 3632 WT, L. reuteri 3632; 3632 VHH3 R1 DC, L. reuteri 3632 delivering ENB‐1D11_R56H with truncated pyrE ; 3632 VHH3c R2 DC, L. reuteri 3632 delivering ENB‐1D11_R56H with intact pyrE ; 3630 WT, L. reuteri 3630; 3630 VHH1 R1 DC, L. reuteri 3630 delivering EAT‐1G4 with truncated pyrE ; 3630 VHH1c R2 DC, L. reuteri 3630 delivering EAT‐1G4 with intact pyrE . Representative results are shown from three independent experiments. VHH3, ENB‐1D11_R56H

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Expressing, Sequencing, Plasmid Preparation, Blocking Assay, Agarose Gel Electrophoresis

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Neutralization of NetB activity by L. reuteri secreted Nbs. (a) A twofold dilution series of precipitated VHH antibodies (5 µM) was preincubated with recombinant NetB, after which 1% chicken erythrocytes was added. The optical density of 100% hemolysis was obtained by diluting the chicken erythrocytes in distilled water. As a control, chicken erythrocytes were incubated with NetB, but without Nbs was used. This resulted in 100% hemolysis (OD 570 = 0.54). A NetB positive control (NetB in PBS) resulted in a mean OD 570 of 0.52 and a PBS negative control (PBS with no NetB and Nbs) yielded a mean OD 570 of 0.05. As the initial amounts of L. reuteri and E. coli purified Nbs used for the assay were different, normalized OD 570 values are shown. (b) Western blot analysis binding of anti‐NetB Nb to NetB in the culture supernatant from different C. perfringens clinical isolates. 1, Ladder; 2, NetB positive control (5 µg); 3, C. perfringens JP1011 overnight culture supernatant (10 µl); 4, C. perfringens JP1011 overnight culture supernatant, 10× concentrated (10 µl); 5, C. perfringens JP1011 mid‐log culture supernatant (10 µl); 6, C. perfringens JP1011 midleg culture supernatant, 10× concentrated (10 µl); 7, C. perfringens CP1‐1 overnight culture supernatant (10 µl). The data represent the mean ± SD of the results of three independent experiments. Nbs, nanobodies; PBS, phosphate‐buffered saline; VHH, Variable domain of the Heavy chain of Heavy chain

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Neutralization, Activity Assay, Recombinant, Control, Incubation, Positive Control, Negative Control, Purification, Western Blot, Binding Assay, Saline

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Efficacy evaluation of L. reuteri candidates delivering nanobodies against NetB and α toxin

Article Snippet: CP1‐1 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Control

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: JP1011 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Cloning, Plasmid Preparation, Marker, Isolation

Journal: MicrobiologyOpen

Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis

doi: 10.1002/mbo3.1270

Figure Lengend Snippet: Neutralization of NetB activity by L. reuteri secreted Nbs. (a) A twofold dilution series of precipitated VHH antibodies (5 µM) was preincubated with recombinant NetB, after which 1% chicken erythrocytes was added. The optical density of 100% hemolysis was obtained by diluting the chicken erythrocytes in distilled water. As a control, chicken erythrocytes were incubated with NetB, but without Nbs was used. This resulted in 100% hemolysis (OD 570 = 0.54). A NetB positive control (NetB in PBS) resulted in a mean OD 570 of 0.52 and a PBS negative control (PBS with no NetB and Nbs) yielded a mean OD 570 of 0.05. As the initial amounts of L. reuteri and E. coli purified Nbs used for the assay were different, normalized OD 570 values are shown. (b) Western blot analysis binding of anti‐NetB Nb to NetB in the culture supernatant from different C. perfringens clinical isolates. 1, Ladder; 2, NetB positive control (5 µg); 3, C. perfringens JP1011 overnight culture supernatant (10 µl); 4, C. perfringens JP1011 overnight culture supernatant, 10× concentrated (10 µl); 5, C. perfringens JP1011 mid‐log culture supernatant (10 µl); 6, C. perfringens JP1011 midleg culture supernatant, 10× concentrated (10 µl); 7, C. perfringens CP1‐1 overnight culture supernatant (10 µl). The data represent the mean ± SD of the results of three independent experiments. Nbs, nanobodies; PBS, phosphate‐buffered saline; VHH, Variable domain of the Heavy chain of Heavy chain

Article Snippet: JP1011 , C. perfringens strain isolated from clinical NE and positive for NetB and α toxin , Elanco Animal Health Inc..

Techniques: Neutralization, Activity Assay, Recombinant, Control, Incubation, Positive Control, Negative Control, Purification, Western Blot, Binding Assay, Saline

Journal: iScience

Article Title: Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis

doi: 10.1016/j.isci.2022.105121

Figure Lengend Snippet: Involvement of the NLRP3 inflammasome in MLKL-mediated host defense WT and Mlkl −/− mice were intramuscularly infected with C. perfringens (1 × 10 7 CFU) for 24 h (A) Representative muscle sections. p -MLKL (IHC) stained brown (upper panel, magnification ×100, lower panel, magnification, ×400). D-4-Amino-3-isoxazolidinone-pretreated WT and Mlkl −/− mice were orally infected with C. perfringens (1 × 10 8 CFU) for 24 h (B and C) Representative duodenum and cecum sections. p -MLKL (IHC) stained brown (upper panel, magnification ×100, lower panel, magnification, ×200). LPS-primed WT and Nlrp3 −/− BMDMs were stimulated with ATP (5mM, 30min), C. perfringens strain ATCC13124, CP1 or CP3 (MOI = 20, 90 min). (D) Cell supernatants and cell extracts immunoblotted for Caspase-1, IL-1β and ASC. GAPDH served as loading controls. (E and F) Culture supernatants were analyzed for IL-1β, IL-18 and TNF-α by ELISA. (G) LDH release was quantified to monitor cell lysis. Data are shown as the percentage of LDH release by Triton X-100 treated control cells. (H) Cell supernatants and cell extracts immunoblotted for Caspase-1, GSDMD, IL-1β, ASC, and GAPDH. (I and J) Culture supernatants were analyzed for IL-1β, IL-18, and TNF-α by ELISA. (K) LDH release was quantified to monitor cell lysis. Data are shown as the percentage of LDH release by Triton X-100 treated control cells. LPS-primed WT and Mlkl −/− BMDMs were stimulated with ATP (5mM, 30min), C. perfringens strain ATCC13124, CP1, or CP3 (MOI = 20, 90 min). (L) Cell supernatants and cell extracts immunoblotted for Caspase-1, GSDMD, IL-1β, ASC, p -MLKL, and GAPDH. (M and N) Culture supernatants were analyzed for IL-1β, IL-18 and TNF-α by ELISA. (O) LDH release was quantified to monitor cell lysis. Data are shown as the percentage of LDH release by Triton X-100 treated control cells. LPS-primed WT, Mlkl −/− and Nlrp3 −/− BMDMs were stimulated with ATP (5mM, 30min), C. perfringens strain ATCC13124, CP1, or CP3 (MOI = 20, 90 min). (P and Q) The cells were fixed, permeabilized, and stained for ASC (green). DAPI was used to label nuclei (blue). magnification, ×400. The percentage of cells containing ASC speckles was quantified. LPS-primed WT and Mlkl −/− BMDMs were stimulated with live bacteria (MOI = 20, 90 min), heat-killed bacteria (HK, MOI = 20, 90 min), bacterial culture supernatants (SN, 5 h) or bacterial α-toxin (20 μg/mL, 5 h). (R and T) Culture supernatants were analyzed for IL-1β, IL-18, and TNF-α by ELISA. (U–W) To determine the bacterial killing capacity of BMDMs, LPS-primed WT, Mlkl −/− , Nlrp3 −/− , or Caspase-1/11 −/− BMDMs were incubated with rIL-18 (1000 pg/mL) or PBS for 1 h before were infected with C. perfringens strain ATCC13124, CP1 or CP3 (MOI = 5) for 6 h, the supernatants were collected and plated on BHI agar plates to enumerate the bacteria after 24 h of anaerobic culture. Graphs are means ± SD from data pooled from four to six (E, F, G, I, J, K, M, N, O, Q, R, S, T, U, V, and W) biological replicates. Data were considered significant when ∗p < 0.05 or ∗∗p < 0.01.

Article Snippet: C. perfringens strain ATCC13124 , Guangdong Huankai Microbial Sci.&Tech.CO.,Ltd , FSCC137003.

Techniques: Infection, Staining, Enzyme-linked Immunosorbent Assay, Lysis, Control, Bacteria, Incubation

Journal: iScience

Article Title: Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis

doi: 10.1016/j.isci.2022.105121

Figure Lengend Snippet: Extracellular traps formation contributes to MLKL-mediated host defense LPS-primed WT and Mlkl −/− BMDMs were infected with C. perfringens strain ATCC13124, CP1, or CP3 (MOI = 20, 90 min). (A) DNA decorated with histone and MPO within the extracellular traps structures was detected by immunofluorescence. Histone (green), MPO (green) and DNA (orange/blue). magnification, ×400. (B) Macrophage extracellular traps were quantified using Fiji and expressed as the percentage of extracellular traps. (C) The formation of bacteria-induced macrophage extracellular traps was quantified using Sytox Green. Cells stimulated with zymosan (1 mg/mL) were used as positive controls. WT and Mlkl −/− mice were intramuscularly infected with C. perfringens (1 × 10 7 CFU) for 24 h (D and E) Representative muscle sections. Histone was analyzed by immunofluorescence (magnification, ×400), and extracellular traps were quantified using Fiji and expressed as the percentage of extracellular traps. D-4-Amino-3-isoxazolidinone-pretreated WT and Mlkl −/− mice were orally infected with C. perfringens (1 × 10 8 CFU) for 24 h (D and E) Representative duodenum and cecum sections. Histone was analyzed by immunofluorescence (magnification, ×200), and extracellular traps were quantified using Fiji and expressed as the percentage of extracellular traps. Graphs are means ± SD from data pooled from four to five (B, C, and E) biological replicates. Data were considered significant when ∗∗p < 0.01. See also Figures S1 and .

Article Snippet: C. perfringens strain ATCC13124 , Guangdong Huankai Microbial Sci.&Tech.CO.,Ltd , FSCC137003.

Techniques: Infection, Immunofluorescence, Bacteria

Journal: iScience

Article Title: Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis

doi: 10.1016/j.isci.2022.105121

Figure Lengend Snippet: Blocking NLRP3 inflammasome signaling attenuates MLKL-mediated extracellular traps formation following C. perfringens challenge LPS-primed WT and Mlkl −/− BMDMs were infected with C. perfringens strain ATCC13124, CP1, or CP3 (MOI = 20, 90 min). (A) Cell extracts immunoblotted for p-P38, P38, p -JNK, JNK, p -ERK1/ERK2, ERK1/ERK2, and GAPDH. Cells stimulated with zymosan (1 mg/mL) were used as positive controls. LPS-primed WT, Mlkl −/− , Nlrp3 −/− , and Caspase-1/11 −/− BMDMs were treated with or without rIL-18 (1000 pg/mL) for 1 h before were infected with C. perfringens strain ATCC13124, CP1 or CP3 (MOI = 20, 90 min). (B) DNA decorated with histone and MPO within the extracellular traps structures were detected by immunofluorescence. Histone (green), MPO (green) and DNA (orange/blue). magnification, ×400. (C) Macrophage extracellular traps were quantified using Fiji and expressed as the percentage of extracellular traps. (D) The formation of bacteria-induced macrophage extracellular traps was quantified using Sytox Green. Cells stimulated with ATP (5 mM, 30 min) were used as positive controls. Graphs are means ± SD from data pooled from four to five (C and D) biological replicates. NT: Untreated cells. Data were considered significant when ∗∗p < 0.01. See also Figure S3 .

Article Snippet: C. perfringens strain ATCC13124 , Guangdong Huankai Microbial Sci.&Tech.CO.,Ltd , FSCC137003.

Techniques: Blocking Assay, Infection, Immunofluorescence, Bacteria

Journal: iScience

Article Title: Mixed lineage kinase-like protein protects against Clostridium perfringens infection by enhancing NLRP3 inflammasome-extracellular traps axis

doi: 10.1016/j.isci.2022.105121

Figure Lengend Snippet:

Article Snippet: C. perfringens strain ATCC13124 , Guangdong Huankai Microbial Sci.&Tech.CO.,Ltd , FSCC137003.

Techniques: Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Isolation, Staining, LDH Cytotoxicity Assay, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: IDCases

Article Title: An autopsy case of gas gangrene, massive intravascular hemolysis, and cytokine storm due to Clostridium perfringens type A infection

doi: 10.1016/j.idcr.2024.e02085

Figure Lengend Snippet: Patient with lethal Clostridium perfringens infection with massive intravascular hemolysis and gas gangrene. (a) Serum of the patient at presentation. (b) Abdominal computed tomography (CT) images obtained at presentation (left panel), 1.5 h (middle panel), and 2.5 h after death (right panel). The lesion in the right lobe of the liver, initially seen as a low-density area at presentation (left panel), was replaced by a gas-filled cavity 1.5 h later (middle panel). A postmortem CT scan revealed rapid and massive expansion of gas-filled cavities in the right and left lobes of the liver (right panel).

Article Snippet: Genomic PCRs targeting CPA, CPB, ETX, ITX, CPE, NetB, PFO, and ColA were performed using bacterial DNA of C. perfringens isolated from the patient’s blood and a commercially available type A strain of C. perfringens (JCM#1290; RIKEN BioResource Research Center, Tsukuba, Japan).

Techniques: Infection, Computed Tomography

Journal: IDCases

Article Title: An autopsy case of gas gangrene, massive intravascular hemolysis, and cytokine storm due to Clostridium perfringens type A infection

doi: 10.1016/j.idcr.2024.e02085

Figure Lengend Snippet: Host cytokine responses against Clostridium perfringens isolated from this case. (a) Toxin profiles of Clostridium perfringens isolated from the patient and a commercially available type A strain (JCM#1290). The leftmost lane represents the 100-bp DNA ladder. Agarose gel electrophoresis of polymerase chain reaction (PCR) products revealed that C. perfringens isolated from the patient expressed CPA, PFO, and ColA, but not CPE. CPA, C. perfringens α toxin; PFO, perfringolysin O; ColA, collagenase. (b) Profiles of serum cytokines and chemokines. Cytokine and chemokine arrays revealed heightened proinflammatory responses in the patient serum. CCL2; C-C chemokine ligand 2, CXCL8; C-X-C motif chemokine ligand 8, G-CSF; granulocyte-colony stimulating factor. (c) Toll-like receptors (TLRs) involved in the production of proinflammatory cytokines by C. perfringens . Splenocytes prepared from C57BL/6 mice or mice deficient in TLR2 and/or TLR4 were stimulated with heat-killed C. perfringens . IL-6 mRNA expression was expressed as the mean ± standard error of the mean. * * P < 0.01.

Article Snippet: Genomic PCRs targeting CPA, CPB, ETX, ITX, CPE, NetB, PFO, and ColA were performed using bacterial DNA of C. perfringens isolated from the patient’s blood and a commercially available type A strain of C. perfringens (JCM#1290; RIKEN BioResource Research Center, Tsukuba, Japan).

Techniques: Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Expressing