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1.
Poult Sci ; 103(6): 103681, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38603932

ABSTRACT

Cellulitis is an important disease in commercial turkey farms associated with significant economic loss. Although the etiology of cellulitis is not fully elucidated, Clostridium septicum (C. septicum) is one of the main causes of this infectious disease. In this study, we report the development of a quantitative real-time PCR (qRT PCR) assay targeting the alpha-toxin gene (csa), which involves a prior 15-cyle PCR using a nested pair of primers to increase the detection sensitivity. Additionally, the TaqMan probe was employed to increase the target-specificity of the assay. The performance of our nested qRT-PCR assay was evaluated using Clostridium isolates from turkey farms, representing both septicum and non-septicum species, as well as sponge swab samples from turkey farms. Our step-by-step development of the assay showed that the csa gene is a suitable target for specific detection of C. septicum strains and that the inclusion of nested PCR step significantly increased the detection sensitivity of the final qRT PCR assay. The performance of the assay was also validated by a high correlation of the threshold cycle numbers of the qRT PCR assay with the relative abundance of C. septicum read counts in 16S rRNA gene microbiota profiles of the C. septicum-containing samples from turkey farms.


Subject(s)
Clostridium Infections , Clostridium septicum , Poultry Diseases , Real-Time Polymerase Chain Reaction , Turkeys , Real-Time Polymerase Chain Reaction/veterinary , Real-Time Polymerase Chain Reaction/methods , Animals , Turkeys/microbiology , Clostridium Infections/veterinary , Clostridium Infections/microbiology , Clostridium Infections/diagnosis , Clostridium septicum/isolation & purification , Clostridium septicum/genetics , Poultry Diseases/microbiology , Poultry Diseases/diagnosis , Sensitivity and Specificity , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/analysis
2.
BMC Vet Res ; 19(1): 8, 2023 Jan 13.
Article in English | MEDLINE | ID: mdl-36639759

ABSTRACT

BACKGROUND: The pathogenic Clostridia cause neurotoxic, histotoxic and enterotoxic infections in humans and animals. Several Clostridium species have been associated with abomasitis in ruminants. The present study aimed to investigate the frequency, and the presence of virulence genes, of Clostridium perfringens, Paeniclostridium sordellii and Clostridium septicum in lambs and goat kids with hemorrhagic abomasitis. RESULTS: A total of 38 abomasum samples, collected from lambs and goat kids of 1 week to 1 month of age in different farms located in eastern Turkey between 2021 and 2022, were evaluated by histopathology, culture and PCR. At necropsy, the abomasum of the animals was excessively filled with caseinized content and gas, and the abomasum mucosa was hemorrhagic in varying degrees. In histopathological evaluation, acute necrotizing hemorrhagic inflammation was noted in abomasum samples. The examination of swab samples by culture and PCR revealed that C. perfringens type A was the most frequently detected species (86.84%) either alone or in combination with other Clostridium species. P. sordellii, C. perfringens type F and C. septicum were also harboured in the samples, albeit at low rates. Beta2 toxin gene (cpb2) was found in three of C. perfringens type A positive samples. CONCLUSION: It was suggested that vaccination of pregnant animals with toxoid vaccines would be beneficial in terms of protecting newborn animals against Clostridial infections. This study investigated the presence of clostridial toxin genes in abomasal samples for the first time in Turkey.


Subject(s)
Clostridium Infections , Gastritis , Goat Diseases , Sheep Diseases , Animals , Clostridium Infections/epidemiology , Clostridium Infections/microbiology , Clostridium Infections/veterinary , Clostridium perfringens/genetics , Clostridium septicum/genetics , Clostridium sordellii , Gastritis/epidemiology , Gastritis/microbiology , Gastritis/veterinary , Goat Diseases/epidemiology , Goat Diseases/microbiology , Goats , Hemorrhage/veterinary , Sheep , Sheep Diseases/epidemiology , Sheep Diseases/microbiology , Sheep, Domestic , Turkey/epidemiology
3.
Anaerobe ; 71: 102406, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34214691

ABSTRACT

Clostridium septicum endophthalmitis is an extremely rare infection with only a few cases reported in the literature. It has an endogenous origin and is associated with gastrointestinal and haematological malignancies. We present the case of a 62-year-old male who presented this infection as the first manifestation of a colon adenocarcinoma.


Subject(s)
Clostridium Infections/microbiology , Clostridium septicum/isolation & purification , Colonic Neoplasms/complications , Endophthalmitis/microbiology , Clostridium Infections/diagnosis , Clostridium septicum/genetics , Clostridium septicum/physiology , Endophthalmitis/etiology , Humans , Male , Middle Aged
4.
Anaerobe ; 32: 34-36, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25481351

ABSTRACT

Clostridium septicum is an uncommon cause of severe infection. Real-time PCR against the C. septicum-specific alpha-toxin gene (csa) was used to estimate the prevalence of this microbe in human stool from 161 asymptomatic community-dwelling adults and 192 hospitalized patients with diarrhea. All samples were negative, suggesting a low prevalence.


Subject(s)
Carrier State , Clostridium Infections/epidemiology , Clostridium Infections/microbiology , Clostridium septicum/genetics , Feces/microbiology , Adult , Clostridium Infections/diagnosis , Clostridium septicum/classification , DNA, Bacterial , Gas Gangrene/epidemiology , Gas Gangrene/microbiology , Genes, Bacterial , Humans , Polymerase Chain Reaction , Prevalence
5.
Microbiol Immunol ; 57(3): 163-9, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23278518

ABSTRACT

Clostridium septicum alpha-toxin has a unique tryptophan-rich region ((302)NGYSEWDWKWV(312)) that consists of 11 amino acid residues near the C-terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan-rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)-anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI-anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan-rich region plays an important role in the cytotoxic mechanism of alpha-toxin, especially in the binding to GPI-anchored proteins as cell receptors.


Subject(s)
Bacterial Toxins/metabolism , Bacterial Toxins/toxicity , Clostridium septicum/metabolism , Glycosylphosphatidylinositols/metabolism , Proteins/metabolism , Tryptophan/metabolism , Animals , Bacterial Toxins/genetics , Cell Survival/drug effects , Chlorocebus aethiops , Clostridium septicum/genetics , DNA Mutational Analysis , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutant Proteins/toxicity , Protein Binding , Tryptophan/genetics , Vero Cells
6.
Anaerobe ; 18(5): 504-7, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22975141

ABSTRACT

Clostridial myositis is an acute, generally fatal toxemia that is considered to be rare in pet animals. The present report describes an unusual canine clostridial myositis that was diagnosed by a new multiplex-PCR (mPCR) designed for simultaneous identification of Clostridium sordellii, Clostridium septicum, Clostridium perfringens type A, Clostridium chauvoei, and Clostridium novyi type A. A ten-month-old male Rottweiler dog, that had displayed lameness and swelling of the left limb for 12 h, was admitted to a veterinary hospital. The animal was weak, dyspneic and hyperthermic, and a clinical examination indicated the presence of gas and edema in the limb. Despite emergency treatment, the animal died in only a few minutes. Samples of muscular tissue from the necrotic area were aseptically collected and plated onto defibrinated sheep blood agar (5%) in anaerobic conditions. Colonies suggestive of Clostridium spp. were submitted to testing by multiplex-PCR. Impression smears of the tissues, visualized with Gram and also with panoptic stains, revealed long rod-shaped organisms, and specimens also tested positive using the fluorescent antibody technique (FAT). The FAT and mPCR tests enabled a diagnosis of C. septicum myonecrosis in the dog.


Subject(s)
Clostridium septicum/isolation & purification , Dog Diseases/diagnosis , Dog Diseases/microbiology , Gas Gangrene/veterinary , Molecular Diagnostic Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Animals , Clostridium septicum/genetics , Dogs , Gas Gangrene/diagnosis , Gas Gangrene/microbiology , Male , Myositis/diagnosis , Myositis/microbiology , Myositis/veterinary , Necrosis/diagnosis , Necrosis/microbiology , Necrosis/veterinary
7.
J Microbiol Methods ; 84(2): 307-11, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21182874

ABSTRACT

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.


Subject(s)
Bacteriological Techniques/methods , Clostridium Infections/veterinary , Clostridium chauvoei/isolation & purification , Clostridium septicum/isolation & purification , Polymerase Chain Reaction/methods , Triose-Phosphate Isomerase/genetics , Animals , Cattle , Cattle Diseases/diagnosis , Cattle Diseases/microbiology , Clostridium Infections/diagnosis , Clostridium chauvoei/classification , Clostridium chauvoei/genetics , Clostridium septicum/classification , Clostridium septicum/genetics , DNA Primers/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Goat Diseases/diagnosis , Goat Diseases/microbiology , Goats , Molecular Sequence Data , Oligonucleotide Probes/genetics , Sensitivity and Specificity , Sequence Analysis, DNA , Sheep , Sheep Diseases/diagnosis , Sheep Diseases/microbiology
8.
Mol Cell Probes ; 24(4): 211-8, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20399850

ABSTRACT

Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.


Subject(s)
Chickens/microbiology , Clostridium septicum/genetics , Clostridium septicum/isolation & purification , Dermatitis/veterinary , Gas Gangrene/veterinary , Polymerase Chain Reaction/methods , Poultry Diseases/microbiology , Animals , Biological Assay , Dermatitis/complications , Dermatitis/diagnosis , Dermatitis/microbiology , Gas Gangrene/complications , Gas Gangrene/diagnosis , Gas Gangrene/microbiology , Polymerase Chain Reaction/veterinary , Poultry Diseases/diagnosis , Reference Standards , Sensitivity and Specificity , Time Factors
9.
Mol Cell Probes ; 24(4): 204-10, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20362050

ABSTRACT

Clostridium chauvoei is the causative agent of blackleg in cattle and sheep. The clinical symptoms of this severe disease are very similar to that of malignant edema (Clostridium septicum), infections of other Clostridium species belonging to the gas edema complex, and anthrax (Bacillus anthracis). C. chauvoei and C. septicum are closely related taxa and share many phenotypic properties hampering diagnosis by using traditional microbiological methods. Thus, there is a need for a fast and reliable identification method for specific detection of both species in clinical samples. The multiplex real-time PCR assay presented here is based on the detection of the spo0A gene and enables the simultaneous identification of C. chauvoei and C. septicum. The assay design includes an amplification control DNA template for the recognition of PCR-inhibitors. Assay validation was performed using a collection of 29 C. chauvoei, 38 C. septicum strains and 26 strains of other Clostridium species. Furthermore, the real-time PCR assay was successfully tested on tissue samples from 19 clinical blackleg cases. The assay allowed the reliable detection of one picogram DNA which represents approximate 239 genome equivalents.


Subject(s)
Clostridium chauvoei/genetics , Clostridium chauvoei/isolation & purification , Clostridium septicum/genetics , Clostridium septicum/isolation & purification , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/veterinary , Animals , Base Sequence , Biological Assay , Cattle , Clostridium Infections/diagnosis , Clostridium Infections/microbiology , Clostridium Infections/veterinary , DNA Primers/metabolism , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Genes, Bacterial/genetics , Hydrolysis , Molecular Sequence Data , Polymerase Chain Reaction/standards , Reference Standards , Sequence Analysis, DNA , Time Factors
10.
Anaerobe ; 15(3): 99-106, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19402197

ABSTRACT

Clostridium septicum is a highly virulent, anaerobic bacterium capable of establishing necrotizing tissue infections and forming heat resistant endospores. Disease is primarily facilitated by secretion of numerous toxic products including a lethal pore-forming cytolysin. Spontaneously occurring clostridial myonecrosis involving C. septicum has recently reemerged as a concern for many poultry producers. However, despite its increasing prevalence, the epidemiology of infection and population structure of C. septicum remains largely unknown. In this study a multilocus sequence typing (MLST) approach was utilized to examine evolutionary relationships within a diverse collection of C. septicum isolates recovered from poultry flocks experiencing episodes of gangrenous dermatitis. The 109 isolates examined represented 42 turkey flocks and 24 different flocks of broiler chickens as well as C. septicum type strain, ATCC 12464. Isolates were recovered predominantly from gangrenous lesions although isolates from livers, gastrointestinal tracts, spleens and blood were included. The loci analyzed were csa, the major lethal toxin produced by C. septicum, and the housekeeping genes gyrA, groEL, dnaK, recA, tpi, ddl, colA and glpK. These loci were included in part because of their previous use in MLST analysis of Clostridium perfringens and Clostridium difficile. Results indicated a high level of conservation present within these housekeeping gene fragments when compared to what has been previously reported for the aforementioned clostridia. Of the 5352 bp of sequence data examined for each isolate, 99.7% (5335/5352) was absolutely conserved among the 109 isolates. Only one of the ten unique sequence types, or allelic profiles, identified among the isolates was recovered from both turkeys and broiler chickens suggesting some host species preference. Phylogenetic analyses identified two unique clusters, or clonal complexes, among these poultry isolates which may have important epidemiological implications for poultry producers in the United States. This work indicates a predominantly clonal population structure for C. septicum although some evidence of recombination was also observed.


Subject(s)
Chickens/microbiology , Clostridium Infections/veterinary , Clostridium septicum/classification , Clostridium septicum/genetics , Poultry Diseases/microbiology , Turkeys/microbiology , Animals , Bacterial Typing Techniques , Clostridium Infections/microbiology , Clostridium septicum/isolation & purification , Cluster Analysis , DNA Fingerprinting , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genotype , Molecular Epidemiology , Phylogeny , Sequence Analysis, DNA , Sequence Homology , United States
11.
Microbes Infect ; 11(3): 413-8, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19284973

ABSTRACT

Clostridium perfringens and Clostridium septicum are the most common causes of clostridial myonecrosis or gas gangrene. Although they mediate a similar disease pathology, they elaborate functionally very different alpha-toxins. We used a reciprocal complementation approach to assess the contribution of the primary toxin of each species to disease and found that C. perfringens alpha-toxin (PLC) was able to mediate the gross pathology of myonecrosis even in a C. septicum background, although it could not induce vascular leukostasis. Conversely, while C. septicum alpha-toxin restored some virulence to a C. perfringens plc mutant, it was less active than in its native background.


Subject(s)
Bacterial Toxins/genetics , Bacterial Toxins/toxicity , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/toxicity , Clostridium perfringens/pathogenicity , Clostridium septicum/pathogenicity , Gas Gangrene/microbiology , Type C Phospholipases/genetics , Type C Phospholipases/toxicity , Animals , Clostridium perfringens/genetics , Clostridium septicum/genetics , Female , Genetic Complementation Test , Mice , Mice, Inbred BALB C
12.
Sheng Wu Gong Cheng Xue Bao ; 23(1): 67-72, 2007 Jan.
Article in Chinese | MEDLINE | ID: mdl-17366890

ABSTRACT

In order to amplify alpha toxin gene of Clostridium septicum HeB01 strain, one pair of primers was designed according to the GenBank sequence, and a 1323bp alpha toxin gene fragment was obstained by PCR. Sequence analysis indicated that the homology of the nucleotid sequence of HeB01 strain to those other reference strains was more than 99.5% . The expression plasmid pQE30-alpha was constructed by inserting alpha toxin gene into the prokaryotic expression vector pQE30. The plasmid expressed when the recombinant strain M15(pQE30-alpha) was induced by IPTG. The specific 48 kD protein was detected SDS-PAGE and the immunogenicity of the expressed alpha toxin was confirmed by Western blot and ELISA. The expressed alpha toxin was transformed into alpha toxoid vaccine by adding 0.3% formaldehyde into alpha toxin. The protective immune response was proved after the mice was immunized with alpha toxoid vaccine. The results showed that the recombinanted strain M15 (pQE30-alpha) could be as a candidate of alpha toxoid vaccine to provide protective immune response against clostridium septicum infection.


Subject(s)
Bacterial Toxins/immunology , Clostridium Infections/immunology , Clostridium septicum/immunology , Toxoids/immunology , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Blotting, Western , Cloning, Molecular , Clostridium Infections/microbiology , Clostridium Infections/prevention & control , Clostridium septicum/genetics , Clostridium septicum/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Mice , Polymerase Chain Reaction , Recombinant Proteins/administration & dosage , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Vaccination
13.
Biochemistry ; 45(48): 14347-54, 2006 Dec 05.
Article in English | MEDLINE | ID: mdl-17128973

ABSTRACT

Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin that belongs to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule on the basis of its functional and sequence similarity with aerolysin, for which the crystal structure has been determined. In this study, we have substituted the entire primary structure of AT with alanine or cysteine to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization, and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane beta-hairpin of the prepore oligomer.


Subject(s)
Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Clostridium septicum/chemistry , Clostridium septicum/metabolism , Amino Acid Sequence , Bacterial Toxins/genetics , Binding Sites , Clostridium septicum/genetics , Conserved Sequence , Crystallography, X-Ray , Cysteine/genetics , Cysteine/metabolism , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Protein Structure, Tertiary , Sequence Alignment , Structural Homology, Protein
14.
Vet Microbiol ; 114(1-2): 51-9, 2006 Apr 16.
Article in English | MEDLINE | ID: mdl-16337096

ABSTRACT

Clostridium septicum alpha-toxin genes were sequenced with the polymerase chain reaction (PCR) products amplified from DNAs of 25 C. septicum strains, and were classified into 10 patterns. Alpha-toxins were purified from the culture supernatant of four C. septicum strains (strains No. 44, Kagoshima 8, Mie and Tokachi) which were specially chosen from patterns of the deduced amino acid sequences. The molecular weights of the alpha-toxins were not different according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. However, the isoelectric points between the alpha-toxins of No. 44 and Tokachi strains differed markedly. Cross-neutralization tests were performed with purified alpha-toxins and antitoxins in mice and in Vero cells. Each antitoxin showed roughly the same titers against the four alpha-toxins in mice and completely identical titers against these in Vero cells. Calves immunized with toxoid prepared from the culture supernatant of No.44 strain were challenged by exposure to spores of Mie strain. The toxoid conferred protection against the challenge in calves. From these results, although genetic variation has been observed within the C. septicum alpha-toxin gene, C. septicum strains toxoid of strain No.44 induces protective immunity against exposure to C. septicum that produce other subtypes of alpha-toxin containing several different amino acid residues.


Subject(s)
Bacterial Toxins/genetics , Bacterial Toxins/immunology , Clostridium septicum/genetics , Genetic Variation/genetics , Amino Acid Sequence , Animals , Antitoxins , Bacterial Toxins/chemistry , Bacterial Toxins/toxicity , Base Sequence , Cattle , Chlorocebus aethiops , Clostridium septicum/classification , Clostridium septicum/immunology , Cross Reactions , DNA Primers/chemistry , Lethal Dose 50 , Male , Mice , Molecular Sequence Data , Neutralization Tests/methods , Phylogeny , Polymerase Chain Reaction/methods , Sequence Analysis, DNA , Toxoids/immunology , Vero Cells
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